Di-isopropyl-phosphinoyl-alkanes as topical agents for the treatment of sensory discomfort

ABSTRACT

The present discovery pertains generally to the field of therapeutic compounds. More specifically the present discovery pertains to certain di-isopropyl-phosphinoyl-alkanes as described herein, DIPA-1-5, DIPA-1-6, DIPA-1-7, DIPA-1-8, and DIPA-1-9, collectively referred to herein as “DIPA compounds”, that are useful, for example, in the treatment of disorders (e.g., diseases) including: sensory discomfort (e.g., caused by irritation, itch, or pain); a skin dysesthesia; dermatitis; ocular pain and discomfort; heat discomfort; heat stress; flushing and/or night sweats (vasomotor symptoms); post-operative hypothermia; post-anaesthetic shivering; nasal congestion and nasal obstruction; pharyngeal and esophageal discomfort; fatigue; tiredness; depression; cognitive dysfunction; and to enhance cognitive function. The applicant has found that localized delivery of DIPA compounds to the upper eyelid and/or facial skin has an alerting and enhancement effect on behavior and can be used to give a cosmetic refreshing look, to mental alertness, to reduce fatigue, and to improve work output. The present discovery also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, for example, in therapy, in diagnosis of neuropathic pain, and in study of TRPM8 function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of US 2015/0164924 A1,published Jun. 18, 2015.

BACKGROUND OF THE INVENTION

Field of the Invention

The present discovery pertains generally to the field of therapeuticcompounds. More specifically the present discovery pertains to certaindi-isopropyl-phosphinoyl-alkanes as described herein (DIPA-5, DIPA-1-6,DIPA-1-7, DIPA-1-8, and DIPA-1-9, collectively referred to herein as“DIPA compounds”) that are useful, for example, in the treatment ofdisorders (e.g., diseases) including: sensory discomfort (e.g., causedby irritation, itch, or pain); a skin dysesthesia; atopic dermatitis;ocular pain and discomfort; heat discomfort; heat stress; flushingand/or night sweats (vasomotor symptoms); post-operative hypothermia;post-anaesthetic shivering; fatigue; tiredness; depression; cognitivedysfunction; and to enhance cognitive function. The present inventionalso pertains to pharmaceutical compositions comprising such compounds,and the use of such compounds and compositions, for example, in therapy,diagnosis, and laboratory investigations. The unusual property of theDIPA molecules is the ability to penetrate the keratinized layers of theskin [stratum corneum] to reach receptive targets underneath.

Description of Related Art

Heat abstraction from the body's surfaces can refresh the senses,relieve discomfort, attenuate pain, and suppress inflammation.Abstraction of heat from body surfaces evokes sensations that are termedcool, refreshing cool, chilly, cold, icy cold and painful cold. Forexample, air blown onto the face from a fan or an air-conditioner canreduce tiredness and increase alertness. A wet towel applied to theforehead can relieve discomfort from a fever or a headache. The methodsof heat abstraction, with gas, liquids, or solids, achieve their effectsby physically lowering tissue temperatures and by activating signals tothe brain.

Chemical sensory/cooling agents are molecules that can mimic thesensations of heat abstraction without a change in tissue temperatures.The exact sensations produced by chemicals depend on the selection ofthe active ingredient and the site and method of delivery. With most ofthe agents currently in use, some degree of chemical cooling on thescalp and skin of the face, nostrils, and philtrum can be elicited, butthe effects do not last long. For the skin of the torso and limbs overtsensations of coolness and cold are more difficult to elicit andsustain. The skin has a keratinized layer of dead cells, called thestratum corneum, that is a formidable barrier to drug penetration intothe epidermis and dermis. The neuronal receptive fields to detecttemperature changes are located in the skin sub-layers.

The term “chemical cooling agent” can be ambiguous because, for example,chemicals such as ethyl chloride as a gas, ethanol as a liquid, liquidnitrogen, or carbon dioxide as a solid, applied to the skin can evokeheat abstraction sensations by reducing tissue temperatures. In thisapplication, chemical cooling agents will refer only to agents thatelicit sensations of heat abstraction without a lowering of tissuetemperatures.

The inventor has previously identified several p-menthane carboxamidecompounds that, when applied to the philtrum skin, simulate effects ofheat abstraction for >1.5 hr without decreasing tissue temperature (Wei,E T. Sensory/cooling agents for skin discomfort. Journal Skin BarrierResearch 14: 5-12, 2012). These compounds are relativelywater-insoluble.

“Thermal comfort” is a well-developed concept in ergonomics,engineering, and architecture. The term refers to a condition in which aperson wearing a normal amount of clothing feels neither too cold nortoo warm. Thermal comfort is important for one's well-being and forproductivity. It is achieved when the air temperature, humidity, and airmovement are within a specified range called the “comfort zone” [24 to27° C. for UK and USA]. It is has been known for some time that anenvironmental temperature below 21.1° C. (70° F.) is optimal for workperformance, and that the best temperature is in the range of 18.3 to20° C. (65 to 68° F.) (Dawson et al., 2009, “Nine switches of humanalertness”, www.circadian.com, presentation from Circadian Technologies,Inc., Houston, Tex., USA, October 2009]. Experimentally, an improvementin performance can be demonstrated at 20° C. versus a 23° C. environment[Tham and Willem, Room air temperatures affects occupant's physiology,perceptions, and mental alertness. Building Environment 45: 40-44,2010]. Conversely, careful studies have documented that work performanceand productivity (output/input) drop by 2% for every increment of +1° C.above 25° C. up to 33° C. [Tanabe et al. Indoor environmental qualityand productivity. Rehva Journal (Federation of the European Heating andAir Conditioning Associations) June, 2007]

The site where temperature is detected on the skin qualitatively affectsperception of thermal comfort. Temperature sensitivity over the bodysurface varies over ˜100 fold. The face, especially area around the eyes[periorbital] and mouth [perioral] are very sensitive, but theextremities have poor sensitivity, and the rest of the body isintermediate [Stevens et al. Temperature sensitivity of the body surfaceover the life span. Somatosensory Motor Research 15: 13-28, 1998]. Thesensory innervation of the periorbital and perioral regions are mediatedby the V1 and V2 branches of the trigeminal nerve [5^(th) cranialnerve].

In hot conditions, humans prefer a cool face, and in cold they prefer awarm abdomen. The human brain is particularly susceptible to heat damageand can only tolerate ˜40.5° C., while organs of the torso tolerate >42°C. Thus, a hot face would further heat an overheated brain, andpreference for a low facial temperature in the heat has survival value[Nakamura et al. Relative importance of different surface regions forthermal comfort in humans. Eur. J. Applied Physiology, 113, 63-76,2012]. Alleviating the sense of heat on the face can reduce discomfortand improve an organism's ongoing behavior. Facial cooling can alsoalert the organism and increase vigilance to focus on threats to itswell-being. This fact is well-known to skiers and to people who live incountries with frigid winters.

Known Phosphine Oxides

The 1-dialkyl-phosphinoyl-alkanes [e.g. total number of carbons≦16] aresolvent-like molecules that require only several [1 to 3] steps forsynthesis. They are also known as trialkylphosphine oxides, but thepreferred term now is dialkyl-phosphinoyl-alkane [DAPA]. If two of thealkyl groups are isopropyl, the DAPA could be abbreviated as DIPA[diisopropyl-phosphinoyl-alkane],

Rowsell and Spring [Phosphine oxides having a physiological coolingeffect. U.S. Pat. No. 4,070,496. Jan. 24, 1978], describes a range ofphosphine oxides which have a physiological cooling effect on skin andon the mucous membranes of the body, particularly the nose, mouth,throat and gastrointestinal tract. See, e.g., the table in columns 3 and4 therein. Ten (10) of the compounds shown therein (Table 1) have oneisopropyl group (shown as iso-C₃H₇). None of the compounds has twoisopropyl group₃.

TABLE 1 Compounds in Rowsell et al., 1978 P(═O)R₁R₂R₃ # R₁ R₂ R₃ 2n-C₇H₁₅ iso-C₃H₇ sec-C₄H₉ 3 n-C₈H₁₇ iso-C₃H₇ sec-C₄H₉ 7 n-C₆H₁₃ iso-C₃H₇sec-C₄H₉ 8 n-C₆H₁₃ iso-C₃H₇ cyclo-C₅H₉ 11 n-C₇H₁₅ iso-C₃H₇ cyclo-C₅H₉ 12n-C₆H₁₃ iso-C₃H₇ iso-C₅H₁₁ 15 n-C₇H₁₅ iso-C₃H₇ iso-C₅H₁₁ 26 n-C₆H₁₃iso-C₃H₇ n-C₆H₁₃ 30 n-C₈H₁₇ iso-C₃H₇ cyclo-C₅H₉ 47 iso-C₃H₇ n-C₄H₉(n-C₄H₉)(C₂H₅)CHCH₂

Wei, E. T. [Ophthalmic compositions and method for treating eyediscomfort and pain. US 2005/0059639 A1, Mar. 17, 2005] describes theuse of certain phosphine oxides and the treatment of eye discomfort bythe administration of eye drops containing those compounds. See, e.g.,Table 1 on page 4 therein. Five (5) of the compounds shown therein(Table 2) have one isopropyl group (shown as iso-C₃H₇).

TABLE 2 Compounds in Wei, 2005 P(═O)R₁R₂R₃ # R₁ R₂ R₃ 14 n-C₆H₁₄iso-C₅H₁₁ iso-C₃H₇ 15 n-C₇H₁₅ iso-C₅H₁₁ iso-C₃H₇ 17 n-C₆H₁₄ iso-C₃H₇sec-C₄H₉ 18 n-C₇H₁₅ iso-C₃H₇ sec-C₄H₉ 19 n-C₈H₁₇ iso-C₃H₇ sec-C₄H₉

Siddall et al. [Simplified preparation of some trisubstituted phosphineoxides. J. Chemical Engineering Data 10: 303-305, 1965] reported thesynthesis of 1-di-isopropyl-octane [DIPA-1-8]. Unlike the Tables 1 and 2compounds, the DIPA-1-8 compound has two isopropyl groups. No reports onany bioactivity of such di-isopropyl compounds have previously beenmade.

BRIEF SUMMARY OF THE INVENTION

In this discovery, it was found that application of a specific coolingagent to the periorbital area, especially to the skin of the eyelids,evoked a “dynamic cool” sensation that will arouse an organism andcounteract tiredness. This change in mind-set is akin to achemically-induced anti-fatigue agent such as caffeine. The subjectbecomes more alert and vigilant. The mode of drug delivery is that of atopical skin product, and not that of an ophthalmic product.

In another aspect of this discovery, a chemical has been created andidentified as producing sensations of heat abstraction and penetratingkeratinized tissues. The skin is a frequent site of injury. Inflammationis the response of tissues to injury and the cardinal signs ofinflammation are a feeling of heat [calor] at the site of injury,redness [rubor], swelling [tumor] and pain [dolor] in and around theinjured tissues. The newly synthesized molecule may have value in therelief of the discomfort signs of irritation, itch, and pain frominflamed skin.

In another aspect of this discovery, the potent cooling agentsynthesized that penetrates the skin to elicit cold [e.g.,1-di-isopropyl-phosphinoyl-heptane] may be used as a diagnostic tool todistinguish between pain of neuropathic versus somatic origin. Coldallodynia and hyperalgesia to cold are conditions frequently associatedwith neuropathic pain. In neuropathic pain application of a molecule ofthis discovery will be expected to exacerbate pain, but an oppositeeffect is expected in somatic pain. This differential action may be useddiagnosis.

In another aspect of this discovery, the selective actions of the newmolecules on TRPM8 were characterized. The current laboratory tools forthe study of TRPM8 function are limited, and most investigators usementhol and icilin. The new molecules of this invention may be used as aset of more selective reagents for the study of the physiology andpharmacology of TRPM8 function.

One aspect of the invention pertains to certaindi-isopropyl-phosphinoyl-alkanes described herein (collectively referredto herein as “DIPA compounds”) and particularly compositions thereof andarticles including such DIPA compounds such a pharmaceutical compositioncomprising one or more DIPA compound, as described herein, and apharmaceutically acceptable carrier or diluent. Particularly preferredembodiments include one or more DIPA compounds and a delivery agentcarrying the one or more compounds, where, the delivery agent issuitable for topical delivery

Another aspect of the present discovery pertains to a DIPA compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy, for example, for use a method of treatment of adisorder (e.g., a disease) as described herein.

Another aspect of the present discovery pertains to a kit comprising (a)a DIPA compound, as described herein, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the compound.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is an illustration of a human head, showing facial sites fortesting: (a) infraorbital, (b) buccal cheek, (c) zygomatic, (d)parotid-masseteric cheek, (e) frontal, and (f) periorbital. Sensationsfrom the periorbital and frontal skin is transmitted via the ophthalmicbranch of the trigeminal nerve [V1]. Diagram adapted from Pilsl et al.[Anatomy of the cheek: implications for soft tissue augmentation.Dermatologic surgery: official publication for American Society forDermatologic Surgery 38, 1254-62, 2012].

FIG. 2 shows the strategy for “eyeball cooling” in a subject. A DIPAcompound of this invention is applied to the eyelid skin with the eyesclosed. Contact of the compound to pain fibers of the cornea is avoided.The sensations from the eyelid skin and from the eyeball surface aretransmitted via the ophthalmic branch of the trigeminal nerve. When thenerve fibers are activated, the brain perceives the whole orbit as beingcooled but without topographical specificity. Thus, the brain isenergized by “eyeball cooling”, but without pain or stinging from theeye surface.

FIG. 3 is a graph of response (Relative Fluorescence Units; % ofmaximum) as a function of the logarithm of the concentration of the testcompound (denoted agonist), expressed in .mu.M, for each of embodimentsof the invention: DIPA-1-5 (circles), DIPA-1-6 (squares), DIPA-1-7(inverted triangle), DIPA-1-8 (diamonds), or DIPA-1-9 (up-righttriangle).

FIG. 4 shows chart traces that illustrate, in the first trace (FIG. 4A“Wild Type”), the inhibition of capsaicin-induced depolarization of theisolated mouse vagus by the DIPA-1-7 embodiment, superfused at aconcentration of 1 mg/mL, and, in the second trace (FIG. 4B “TRPM8 KO”),the significant absence of inhibition in the isolated TRPM8 KO(knockout) mouse vagus by DIPA-1-7, superfused at a concentration of 1mg/mL.

FIG. 5 shows the method for measuring the transdermal activity of theDIPA-embodiment compounds applied 20 .mu.L with a micropipette to thecenter of a circle enclosed by cream on the abdominal skin of ananesthetized rat. Shaking frequency was counted for 1 hr after topicalapplication.

FIG. 6 shows shaking frequency [black bars, left ordinate] aftertransdermal application and the lack of correlation to TRPM8 potency[right ordinate, inverse of EC50, potency relative to menthol] for testchemicals. It can be seen that the embodiments DIPA-1-5, DIPA-1-6, andDIPA-1-7 readily evoke robust shaking, but this is not seen with theother analogs.

FIG. 7 shows shaking frequency [black bars, left ordinate] afterintravenous (i.v.) injection of DIPA-1-7 and 2-6 at 1 mg/kg intopentobarbital-anesthetized rats (N=6 per compound). Two trials wereconducted in the same animal and shaking frequency was counted for 30min.

DETAILED DESCRIPTION OF THE INVENTION

In this discovery, new water-soluble compounds [e.g.,1-di-isopropyl-phosphinoyl-heptane] have been identified that potently[<5 mg per dose] and rapidly produce on skin robust and intense coolingsensations. This type of drug action is unusual and has not beenpreviously recognized to be achievable on keratinized surfaces and haslead to new applications as described herein.

Thus the present discovery relates to certain compounds (the DIPAcompounds described herein) which, when delivered onto the skin,especially on periorbital facial skin, selectively and potently evokesensations of “dynamic cool” for at least several hours. The dynamiccool can be repeated without significant diminution of the effects andcan be sustained for the whole day. The sensations on the facial skin donot interfere with the individual's ability to fall asleep. The DIPAcompounds may be used to counteract fatigue and enhance cognitivefunction. The DIPA compounds are administered topically, onto the eyelidskin and avoiding the cornea, and so effects are achieved without directinvasion of brain chemistry. The unusual, surprising feature of the DIPAcompounds, relative to structurally similar analogs, is the ability topermeate through keratinized skin to produce strong sensations. This canbe shown in laboratory animal experiments. Thus, these compounds alsohave applications in the treatment of skin discomfort, especially skinirritation, itch, and pain. The DIPA compounds may be used as selectivelaboratory reagents for the study of TRPM8 function, physiology, andpharmacology. In particular, the novel DIPA-1-7 embodiment may be usedas a diagnostic agent to distinguish pain of neuropathic versus somaticorigin.

DIPA Compounds

The compounds of the present discovery are examples of phosphine oxides,and more particularly, are examples of di-alkyl-phosphinoyl-alkanes[(O═)PR₁R₂R₃] wherein each of R₁, R₂, and R₃ is an alkyl group, and inparticular where R₁ and R₂ are isopropyl, and R₃ is a linear alkyl groupof 5 to 9 carbons and which have the following general formula ofFormula 1:

More specifically, the compounds of the present discovery are shown inTable 3 and collectively referred to herein asdi(isopropyl)-phosphinoyl-alkanes [DIPA] or “DIPA compounds”.

TABLE 3 DIPA-Compounds Formula/ Code Chemical Name Weight ChemicalStructure DIPA- 1-5 1-di(isopropyl)- phosphinoyl- pentane C₁₁H₂₅OP240.32

DIPA- 1-6 1-di(isopropyl)- phosphinoyl- hexane C₁₂H₂₇OP 218.32

DIPA- 1-7 1-di(isopropyl)- phosphinoyl- heptane C₁₃H₂₉OP 232.34

DIPA- 1-8 1-di(isopropyl) phosphinoyl- octane C₁₄H₃₁OP 246.37

DIPA- 1-9 1-di(isopropyl) phosphinoyl- nonane C₁₅H₃₃OP 260.40

Chemical Synthesis

The DIPA compounds were prepared by the following general method: 100 mL(23.7 g, ˜200 mmol) of isopropylmagnesium chloride (orsec-butylmagnesium chloride in the case of the di-sec-butyl derivatives)were obtained from Acros, as a 25% solution in tetrahydrofuran (THF) andplaced under nitrogen in a 500 mL flask (with a stir bar).Diethylphosphite solution in THF (from Aldrich, D99234; 8.25 g, 60.6mmol in 50 mL) was added drop-wise. After approximately 30 minutes, thereaction mixture warmed up to boiling. The reaction mixture was stirredfor an extra 30 minutes, followed by a drop-wise addition of theappropriate n-alkyl iodide solution in THF (from TCI; 60 mmol in 20 mL).The reactive mixture was then stirred overnight at room temperature. Thereaction mixture was diluted with water, transferred to a separatoryfunnel, acidified with acetic acid (˜10 mL), and extracted twice withether. The ether layer was washed with water and evaporated (RotaVapBuchi, bath temperature 40° C.). The light brown oil was distilled underhigh vacuum. The final products, verified by mass as determined by massspectrometry, were transparent liquids that were colourless or slightlypale yellow.

Several samples of 1-7 or 1-8 were sent for detailed analysis by GC-MS(NCE Corporation, Pleasanton, Calif., USA, www.nceanalytical.com).Analysis was conducted on an Agilent GC/MS system 6890/5973 equippedwith a TraceGold TG-624 column, with helium as the carrier gas [flowrate: 1.6 mL/min] and the injector port set 220° C. [split ratio 50:1,temperature program: 100 to 240° C.]. The TIC [total ion chromatogram]showed the main components as having a retention time of 18 to 19 min,with the detected peaks accounting for 97.2% of the total area. Similarresults of 97 to 99% purity were obtained with other samples. When gaschromatography [equipped with a flame ionization detector (Dong WhaCorporation, Seoul, Korea)] was used as the analytical system,synthesized compounds were also found to be 97 to 99%chromatographically pure.

The following compounds were prepared by this method where Table 4Acompounds are embodiments of the invention.

TABLE 4A Chemical structures of tested compounds of this discovery. CodeChemical Name Chemical Structure DIPA-1-5 1-di(isopropyl)-phosphinoyl-pentane

DIPA-1-6 1-di(isopropyl)-phosphinoyl- hexane

DIPA-1-7 1-di(isopropyl)-phosphinoyl- heptane

DIPA-1-8 1-di(isopropyl)-phosphinoyl- octane

DIPA-1-9 1-di(isopropyl)-phosphinoyl- nonane

TABLE 4B Chemical structures of test compounds related to thisdiscovery. Code Chemical Name Chemical Structure 2-41-di(sec-butyl)-phosphinoyl- butane

2-5 1-di(sec-butyl)-phosphinoyl- pentane

2-6 1-di(sec-butyl)-phosphinoyl- hexane

2-7 1-di(sec-butyl)-phosphinoyl- heptane

2-8 1-di(sec-butyl)-phosphinoyl- octane

3-1 1-di(iso-butyl)-phosphinoyl- pentane

3-2 1-di(sec-butyl)-phosphinoyl- 3-methyl-butane

Note that the diisopropyl groups of the 1-x series do not have a chiralcenter but each of the sec-butyl groups in compounds of the 2-x serieshas a chiral centre, and that each chiral centre may independently be inthe (R) or (S) configuration. As a consequence, a compound such as 2-6has four possible stereoisomers: two optically active stereoisomers(i.e., R,R and S,S), and two optically inactive meso forms (i.e., R,Sand S,R). Unless otherwise indicated, a reference to compounds of the2-x series is intended to be reference to any one of the fourstereoisomers, and any mixture of any two or more of the fourstereoisomers. The absence of stereoisomers in the 1-x series is anadvantage in drug development over the 2-x series because frequentlyregulations require that each enantiomer be either synthesized orisolated separately and then individually evaluated for toxicologicalactivities.

General Observations

DIPA compounds are colorless liquids with a density less than water.They are soluble in water or saline at up to 20 mg/mL. When DIPAcompounds are applied to the facial skin as an aqueous solution at 1-10mg/mL or a 1% hydrogel there is little irritation. For certain analogs,contacting the periorbital or malar skin with a solution at aconcentration of 1-10 mg/mL produce a sensation of “dynamic cool” thatis felt within one minute after application. A single application canevoke this “energizing” sensation, which can counteract fatigue forseveral hours. DIPA-1-7, especially, has intense dynamic cooling. Themechanisms underlying this “energizing” effect are discussed in detailin this application.

The method of drug delivery is important to achieve the energizingeffect. As shown in FIG. 2, the drug is applied as a 0.5% gel [1.5%carbopol in purified water] to the eyelid skin with the eyes closed,thus avoiding drug contact with the cornea which is densely innervatedwith pain fibers. The sensory nerves of the eyelids and cornea both sendinformation to the brain via the ophthalmic branch of the trigeminalnerve [5^(th) nerve, V1]. Most likely, the brain does not discriminatetopographically between cooling from eyelid skin or from eyeballsurface. The net perceived effect is a “dynamic cool eyeball” and brainarousal. Avoidance of drug contact with the corneal surface is a keyelement of the success of this treatment because the cornea is denselyinnervated with sensory nerve endings for pain. The penetration of theDIPA compound through the stratum corneum of the eyelid skin is a keyfactor for delivery and is illustrated in the laboratory data shown inTable 10 and in FIG. 2.

Periorbital administration of DIPA and related DAPA compounds will leavea residue on the eyelid skin. When the eyelids become wet, for example,by taking a shower or sweating, the residual compound will wash onto thecornea and cause stinging and irritation. This will limit the choice ofthe compound for applications wherein delivery is to the eyelid skin.Among the compounds of Formula 1, DIPA-1-8 and DIPA-1-9 have minimalresidual irritation, and so are especially useful for the longer termtreatment of ocular discomfort. The efficacy of DIPA-1-9 in thetreatment of patients with the “dry eyes syndrome” is demonstrated inCase Study 7. DIPA-1-7 is useful for a shorter term application ofcognitive function.

Both DIPA-1-7 and DIPA-1-8 (and in particular DIPA-1-7) are especiallyuseful for treatment of skin dysesthesias (e.g., skin irritation, itchyskin, or painful skin), heat discomfort, and heat stress. DIPA-1-6 isnot as long-acting as DIPA-1-7, but is absorbed more easily across theskin, and is therefore especially useful for systemic applications,e.g., in the treatment of flushing and/or night sweats (vasomotorsymptoms).

The effects of DIPA versus the di-sec-butyl congeners were strikinglydifferent in laboratory animals [Table 10]. Perioral or topicalapplication of DIPA [DIPA-1-5, DIPA-1-6, DIPA-1-7] elicits vigorousshaking in the whole animal, but this effect is hardly seen with thedi-sec-butyl congeners. This is because DIPA-1-5, DIPA-1-6, and DIPA-1-7are able to penetrate the membrane barriers in the gut and keratinizedskin. The DIPA compounds have pharmacology that is distinct and unusualrelative to the other DAPA analogs.

Compositions

One aspect of the present discovery pertains to a composition (e.g., apharmaceutical composition) comprising a DIPA compound, as describedherein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

Another aspect of the present discovery pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingmixing a DIPA compound, as described herein, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

In one embodiment, the composition comprises the DIPA compound at aconcentration of 0.005-2.0% wt/vol. In one embodiment, the compositionis a liquid or semi-liquid composition (lotion, cream, or ointment), andcomprises the DIPA compound at a concentration of 0.5-20 mg/mL. In oneembodiment, the composition is a liquid composition, and comprises theDIPA compound at a concentration of 1-5 mg/mL. In one embodiment, thecomposition is a liquid composition, and comprises the DIPA compound ata concentration of 5-10 mg/mL. In one embodiment, the composition is aliquid composition, and comprises the DIPA compound at a concentrationof 10-20 mg/mL.

The composition may be provided with suitable packaging and/or in asuitable container. For example, the composition may be provided as aswab, wipe, pad, or towellette (e.g., suitably sealed in a wrap)carrying a DIPA compound or a composition comprising a DIPA compound.Similarly, the composition may be provided as a patch, e.g., acontrolled-release patch, e.g., suitable for application to the skin,e.g., the skin above the supraclavicular fossa or the steronomastoidmuscle. Similarly, the composition may be provided as an aerosolizedspray delivered from a pressurized container. Similarly, the compositionmay be provided in a manually-activated sprayer (e.g., with a suitablesmall orifice) linked to a reservoir containing a DIPA compound or acomposition comprising a DIPA compound, for example, capable ofdelivering an unit volume (e.g., of 0.05 to 0.15 mL), for example, tothe skin surface.

Use in Methods of Therapy

Another aspect of the present discovery pertains to a DIPA compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy, for example, for use a method of treatment of adisorder (e.g., a disease) as described herein.

Use in the Manufacture of Medicaments

Another aspect of the present discovery pertains to use of a DIPAcompound, as described herein, in the manufacture of a medicament, forexample, for use in a method of treatment, for example, for use a methodof treatment of a disorder (e.g., a disease) as described herein. In oneembodiment, the medicament comprises the DIPA compound.

Methods of Treatment

Another aspect of the present discovery pertains to a method oftreatment, for example, a method of treatment of a disorder (e.g., adisease) as described herein, comprising administering to a subject inneed of treatment a therapeutically-effective amount of a DIPA compound,as described herein, preferably in the form of a pharmaceuticalcomposition.

Disorders Treated

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of: sensory discomfort (e.g., caused by irritation, itch, orpain); a skin dysesthesia; atopic dermatitis; ocular pain anddiscomfort; heat discomfort; heat stress; flushing and/or night sweats(vasomotor symptoms) in post-menopausal women; post-operativehypothermia; post-anaesthetic shivering; fatigue; tiredness; depression;cognitive dysfunction; and to enhance cognitive function.

Disorders Treated—Sensory Discomfort Etc.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of sensory discomfort.

The term “sensory discomfort”, as used herein, relates to irritation,itch, pain, or other dysesthesias (abnormal sensations; such as burningsensations, or feeling the presence of a foreign body, or pins andneedles) from the body surfaces. The term implies activation ofnociceptors located on sensory nerve endings of the body. Nociceptorsare stimulated, for example, by high or low temperatures, mechanicalpressure, chemicals (e.g., capsaicin, acidity, pollutants, etc.),injury, inflammation, and inflammatory mediators. A compound, such asDIPA-1-7, that decreases sensory discomfort, can be termed ananti-nociceptive agent.

In one embodiment, the sensory discomfort is irritation, itch, or pain.In one embodiment, the sensory discomfort is caused by a skindysesthesia. In one embodiment, the skin dysesthesia is skin irritation,itchy skin, or painful skin. In one embodiment, the sensory discomfortis caused by atopic dermatitis. In one embodiment, the sensorydiscomfort is caused by canine atopic dermatitis. In one embodiment, thetreatment is treatment of a skin dysesthesia. In one embodiment, theskin dysesthesia is skin irritation, itchy skin, or painful skin. In oneembodiment, the treatment is treatment of atopic dermatitis. In oneembodiment, the treatment is treatment of canine atopic dermatitis. Inone embodiment, the treatment is treatment of ocular discomfort. In oneembodiment, the ocular discomfort is caused by eye strain; eye fatigue;eye surgery; an airborne irritant or pollutant that interacts with theeye surface; extended wear of contact lenses; excessive exposure to thesun; conjunctivitis; or the dry eyes syndrome. In one embodiment, thetreatment is treatment of heat discomfort. In one embodiment, thetreatment is treatment of heat discomfort for the purpose of improvingathletic performance. In one embodiment, the treatment is treatment ofheat stress. In one embodiment, the treatment is treatment of flushingand/or night sweats (vasomotor symptoms) in a post-menopausal woman. Inone embodiment, the treatment is treatment of post-operative hypothermiaor post-anaesthetic shivering. In one embodiment, the treatment istreatment is to convey a sense of refreshment to the skin in a human.

Disorders Treated—Fatigue Etc.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of fatigue, tiredness, or depression. In one embodiment, thetreatment is treatment of fatigue. In one embodiment, the fatigue isfatigue caused by chronic illness, ageing, a neurological dysfunction,or a psychological dysfunction. In one embodiment, the fatigue isfatigue caused by cancer or cancer-related treatment. In one embodiment,the fatigue is fatigue caused by anxiety, depression, heat stress,cognitive dysfunction, excessive physical exertion, or excessive mentalexertion. In one embodiment, the fatigue is fatigue associated with adecreased ability to think, to concentrate, to study, or to performwork.

Disorders Treated—Cognitive Dysfunction Etc.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of cognitive dysfunction. In one embodiment, the treatment istreatment to enhance cognitive function (e.g., in the healthy as well asthe sick). In one embodiment, the enhanced cognitive function isimproved hand-eye coordination in a sport. In one embodiment, theenhanced cognitive function is improved performance in a game of chanceor of mental skills.

Treatment

The term “treatment,” as used herein in the context of treating adisorder, pertains generally to treatment of a human or an animal (e.g.,in veterinary applications), in which some desired therapeutic effect isachieved, for example, the inhibition of the progress of the disorder,and includes a reduction in the rate of progress, a halt in the rate ofprogress, alleviation of symptoms of the disorder, amelioration of thedisorder, and cure of the disorder. Treatment as a prophylactic measure(i.e., prophylaxis) is also included. For example, use with patients whohave not yet developed the disorder, but who are at risk of developingthe disorder, is encompassed by the term “treatment.” Treatment toenhance the basal levels of cognitive or physical performance ofindividuals who are considered normal or healthy is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound, or a material, composition or dosage formcomprising a compound, which is effective for producing some desiredtherapeutic effect, commensurate with a reasonable benefit/risk ratio,when administered in accordance with a desired treatment regimen.

Combination Therapies

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. For example, the compounds describedherein may also be used in combination therapies, e.g., in conjunctionwith other agents.

One aspect of the present discovery pertains to a DIPA compound asdescribed herein, in combination with one or more (e.g., 1, 2, 3, 4,etc.) additional therapeutic agents. The particular combination would beat the discretion of the physician or the pharmacist who would selectdosages using his common general knowledge and dosing regimens known toa skilled practitioner.

Examples of additional therapeutic agents include: an anti-inflammatoryglucocorticosteroid; an analgesic; a sympathomimetic amine decongestant;an anti-histamine; a local anesthetic; an ophthalmic lubricant; asunscreen ingredient; an anti-acne agent; a keratolytic agent; ananti-hemorrhoidal agent; an agent for vulvar itch or discomfort; anantibiotic; a skin moisturizer; or an anti-skin ageing agent.

Kits

One aspect of the discovery pertains to a kit comprising (a) a DIPAcompound as described herein, or a composition comprising a DIPAcompound as described herein, e.g., preferably provided in a suitablecontainer and/or with suitable packaging; and (b) instructions for use,e.g., written instructions on how to administer the compound orcomposition. The written instructions may also include a list ofindications for which the active ingredient is a suitable treatment. Thewritten instructions (e.g., pamphlet or package label) may include thedosage and administration instructions, details of the formulation'scomposition, the clinical pharmacology, drug resistance,pharmacokinetics, absorption, bioavailability, and contraindications.

Methods of Diagnosis

The DIPA compounds described herein may also be used in diagnosis, forexample, diagnosis of allodynia, for example, cold allodynia. Morespecifically, the DIPA compounds may be used as diagnostic agents forthe diagnosis (e.g., differential diagnosis) of cold allodynia.

Allodynia is pain due to a stimulus which does not normally provokepain. For example, temperature and physical stimuli can provokeallodynia, and it often occurs after injury to a site.

A simple diagnostic tool for differentiating neuropathic pain (e.g.,allodynia) from somatic pain is not yet known. A DIPA compound, such asDIPA-1-7, applied to the skin, can be used to provide differentialdiagnosis of, e.g., cold allodynia.

Routes of Administration

The DIPA compound or pharmaceutical composition comprising the DIPAcompound may suitably be administered to a subject topically, forexample, as described herein.

The term “topical application”, as used herein, refers to delivery ontosurfaces of the body in contact with air, which includes the skin, theanogenital surfaces, the transitional epithelial surfaces of the orbit,the lips, the nose, and the anus, and the aerodigestive tract (nasalmembranes, oral cavity, pharyngeal and esophageal surfaces), lowerrespiratory tracts, and the lumen of the gastrointestinal tract.Particularly preferred sites of application are the surfaces innervatedby the trigeminal and glossopharyngeal nerves which include the scalp,facial skin, periorbital skin, lips, nasal and oral cavities, and thethroat. Additional preferred sites are the surfaces of the neck, elbowsand knees, which are frequently associated with the pruritus of atopiceczema and psoriasis. Yet another preferred site is the scalp, which canbe a site of inflammation in psoriasis and seborrheic dermatitis.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment by topical administration. In one embodiment, the treatment istreatment by topical administration to skin. In one embodiment, thetreatment is treatment by topical administration to facial skin. In oneembodiment, the treatment is treatment by topical administration toperiorbital skin, eyelid skin, malar skin, forehead skin, or scalp. Inone embodiment, the treatment is treatment by topical administration toskin surface of the orbit, frontal bone, or zygomatic. In oneembodiment, the treatment is treatment by topical administration to skinsurface of the anus and/or the male or female genitalia. In oneembodiment, the treatment is treatment by topical administration to skinabove the supraclavicular fossa or the steronomastoid muscle.

The Subject/Patient

The subject/patient may be a mammal, for example, a marsupial (e.g.,kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, amouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian(e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine(e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine(e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey(e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan,gibbon), or a human. In one preferred embodiment, the subject/patient isa human.

Formulations

While it is possible for a DIPA compound to be administered alone, it ispreferable to present it as a pharmaceutical formulation (e.g.,composition, preparation, medicament) comprising at least one DIPAcompound, as described herein, together with one or more otherpharmaceutically acceptable ingredients well known to those skilled inthe art, including, but not limited to, pharmaceutically acceptablecarriers, diluents, excipients, adjuvants, fillers, buffers,preservatives, anti-oxidants, lubricants, stabilisers, solubilisers,surfactants (e.g., wetting agents), masking agents, colouring agents,flavouring agents, and sweetening agents. The formulation may furthercomprise other active agents.

Thus, the present discovery further provides pharmaceuticalcompositions, as described above, and methods of making pharmaceuticalcompositions, as described above. If formulated as discrete units (e.g.,swab, wipe, pads, towellettes, gels, lotion, cream, etc.), each unitcontains a predetermined amount (dosage) of the compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

Formulations may suitably be in the form of liquids, solutions (e.g.,aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups,electuaries, mouthwashes, drops, tablets (including, e.g., coatedtablets), granules, powders, lozenges, pastilles, capsules (including,e.g., hard and soft gelatin capsules), cachets, pills, ampoules,boluses, suppositories, pessaries, tinctures, gels, pastes, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols.

Additionally, the DIPA compound may be used as an adjunct in apharmaceutical formulation or cosmetic formulation.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the DIPA compounds, and compositions comprising the DIPAcompounds, can vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects. The selecteddosage level will depend on a variety of factors including, but notlimited to, the activity of the particular DIPA compound, the route ofadministration, the time of administration, the duration of thetreatment, other drugs, compounds, and/or materials used in combination,the severity of the disorder, and the species, sex, age, weight,condition, general health, and prior medical history of the patient. Theamount of DIPA compound and route of administration will ultimately beat the discretion of the physician, pharmacist, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

Targets for Delivery

Epithelial cells line ducts, cavities and surfaces of organs throughoutthe body. When there are two or more layers of epithelia, it is calledstratified epithelium. Historically, stratified epithelia were dividedinto two broad categories: keratinized stratified epithelia, andnon-keratinized stratified epithelia. Keratinized epithelium, such asthe epidermis of the skin, has an exterior layer of dead cells [stratumcorneum] composed of keratin proteins that are tough andwater-impermeable. By contrast, non-keratinizing stratified epitheliaare located on “soft tissues” of the body such as the lining of thenasal and throat cavities and the oesophageal surface. Keratinizingtissues withstand injury better than non-keratinizing tissues.Non-keratinizing epithelial surfaces must be kept moist by glandular(serous and mucous) secretions in order to avoid desiccation.

A layer of keratin is a formidable barrier for drug access to neuronalreceptive fields embedded in tissues underneath the keratin.

There are currently no topical antinociceptive (pain-suppressant)compounds that have strong efficacy on sensory discomfort fromnon-keratinizng stratified epithelium (NKSE). This is especially truefor sensory discomfort from the oral cavity, pharyngeal, and oesophagealsurfaces.

By experiment, it was discovered that the optimal targets for topicaldelivery of an agent to counteract fatigue and achieve maximal sensoryeffects are located on the receptive fields of ophthalmic and maxillarybranches of the trigeminal nerve. The preferred sites on the face areperiorbital zygomatic=infraorbital, labelled (f), (c), and (a),respectively, in FIG. 1. The periorbital site labelled (f) includes theskin of the eyelids.

FIG. 1 is an illustration of a human head, showing facial sites fortesting: (a) infraorbital, (b) buccal cheek, (c) zygomatic, (d)parotid-masseteric cheek, (c) frontal, and (f) periorbital. Taken fromPilsl et al. [Anatomy of the cheek: implications for soft tissueaugmentation. Dermatologic surgery: official publication for AmericanSociety for Dermatologic Surgery 38, 1254-62, 2012].

To counteract fatigue or heat stress, the active ingredient ispreferably delivered to (a), (c), or (f). Alternatively, if the coolingagent is to be used for flushing and/or night sweats (vasomotorsymptoms) in post-menopausal women, it may also be applied to the skinabove the supraclavicular fossa or the chest. To reduce sensorydiscomfort on the skin, the cooling agent may be directly applied to thesites of injury and/or inflammation.

Secondary sites are the skin over the frontal bone and the scalp(labelled (e)), but higher concentrations of cooling agent are requiredfor (e). The other skin sites, namely, buccal cheek, parotid-massetercheek, periauricular, and chin, lack sensitivity, and sites such as thephiltrum, nasal, temporal region, and neck are topographicallyinconvenient for cooling agent delivery. In practice, the cooling agentcan be sprayed or applied (e.g., with a swab or pad or within a gel,lotion, cream or ointment) over the skin of the orbit, the cheekbone(zygomatic), or on the skin beneath the eye, between the cheekbone andnose. The important receptive fields are from the sub-divisions of thetrigeminal nerve, namely, the zygomaticfacial nerve of the maxillarynerve (V2) and the supraorbital and supratrochlear branches of thefrontal nerve (V1).

One unusual feature of DIPA-1-7 and DIPA-1-8 is that they leave areservoir in the skin after application, so that after the initialsensations have dissipated, the dynamic cooling sensation returns whenthe skin is moist again. This feature is especially beneficial for useof DIPA-1-7 and DIPA-1-8 in conditions of elevated environmentaltemperature. When sweating is activated by heat, the sweatre-solubilizes DIPA-1-7 and DIPA-1-8 and enhances and perpetuates thesensory effect. This self-regulating feedback mechanism makes the effectof DIPA-1-7 and DIPA-1-8 more robust, efficacious, and prolonged.

Methods of Delivery

The delivery of the DIPA compounds can be achieved with the compounddissolved in a solid or semi-solid vehicle, e.g., a cream or anointment, or in a liquid vehicle, e.g., in a solution, a hydrogel, alotion, on a swab, wet wipe, or as an aerosolized mist.

An example is the use of a gel as shown in FIG. 2 for delivery onto theeyelid skin.

Gels are semisolid, jelly-like formulations with varying degrees ofviscosity. A gel forms a solid three-dimensional network that spans thevolume of a liquid medium. Gels are made with gelling agents thatcross-link or associate with a liquid phase. Examples of gelling agentsare: cellulose derivatives [methylcellulose, carboxymethylcellulose,hydroxylpropylcellulose; carbomers [Carbopol®910, Carbopol®941];poloxamers [Pluronic®, Tween]; carbomer polymers, and natural polymerssuch as tragacanth, acacia, gelatin, sodium alginate, alginic acid, andxanthan gum. A single-phase system is a gelling agent plus an activeingredient that dissolves [in water] without visible particles and looksclear. A topical gel optimally liquefies when in contact with skin ormucous membranes. The compounds of Formula 1 are attractive for deliveryas gels because they dissolve in water and form a one-phase system attherapeutic concentrations. The methods for formulating topical gels arewell-known to the art and extensively described in such sites inLubrizol.com [a company that manufactures ingredients for cosmetics,personal care, skin care, and eye care].

For a solid or semi-solid vehicle, a preferred concentration of the DIPAcompound is 0.01 to 2.0% wt/vol. Unless otherwise stated, wt/vol ismeasured in units of g/cm³, and so 0.01% wt/vol is obtained from 0.1 mg(0.0001 g) DIPA compound in 1 cm³ of composition; and 2% wt/vol isobtained from 20 mg (0.02 g) DIPA compound in 1 cm³ of composition.

For a liquid vehicle, a preferred delivered volume is 0.05 to 0.15 mL.Such a volume, delivered for example as a spray, does not cause muchresidual liquid at the delivery site, as the liquid is absorbed.

For a liquid vehicle, a preferred concentration of the DIPA compound isin the range of 0.5 to 20 mg/mL. For the orbit, a preferredconcentration is 1 to 5 mg/mL. For the zygomatic and infraorbital skin,a preferred concentration is 5 to 10 mg/mL. For the forehead skin andscalp, a preferred concentration is 10 to 20 mg/mL.

A preferred amount of the DIPA compound delivered at the site ofapplication is 0.01 to 5 mg; for example, 0.1 to 5 mg.

Wiping of the DIPA compound on the target skin can be done withpre-medicated wipes, which are well-known in personal care products, forexample, to wipe a baby's skin after a diaper change, or to removemake-up on the face (e.g., Pond's 6″×8″ (15 cm×20 cm) Clean SweepCleansing and Make-up Remover Towelettes). Usually, these wipes arepackaged as a single-use sealed unit or in a multi-unit dispenser. Forsingle units, suitable wrapper materials are those which are relativelyvapor impermeable, to prevent drying out of the wipe, and able to form a“peelable” seal. Examples of suitable wipe materials for practicing thisdiscovery include polyamide (20% Nylon)-polyester, rayon (70%)-polyester(30%) formed fabric, polypropylene nonwoven, polyethylene terephthalate(PET), polyester polypropylene blends, cotton, or microfibers (syntheticfibers that measure less than one denier or one decitex).

Alternatively, a solution containing a DIPA compound may be supplied ina reservoir bottle with individual applicators, or as a pre-packagedindividual unit. For example, Puritan 803-PCL applicators are idealcotton-tipped applicators attached to a 3-inch (˜7.5 cm) polystyrene rodfor delivery of a DIPA compound onto the periorbital skin. Examples ofhow such applicators can be individually packaged are the SwabDose™ fromUnicep Corporation (1702 Industrial Drive, Sandpoint, Id., USA), and thePro-Swabs from American Empire Manufacturing (3828 Hawthorne Court,Waukegan, Ill., USA). Each applicator tip is saturated by dipping theabsorbent material of the tip (e.g., 40 to 100 mg of cotton) in 0.5 to1.5 mL of an aqueous solution of a DIPA compound and packaged in anindividual container.

For application to the face, the individual is instructed to gentlyapply the cream, lotion, gel, or wet wipe onto, or to spray, to thetarget facial skin with the eyelids shut, or other skin surface(s). Theinstructions for application may include teaching the individual torepeat application, or “topping up”, to ensure that sufficientcomposition is delivered to the target. Once the subject has learnedwhat to expect, the individual can adjust the dosage (e.g., by dabbingat the medial or lateral edges of the orbit), as needed, to achieve thedesired effect. It has been observed that individuals learn how toeffectively apply the cooling agent after one or two trials and do sowithout risks of discomfort (e.g., eye discomfort).

For application to the anogenital skin or other highly sensitivesurfaces, the DIPA compound may be sprayed with a hand-activated manualpump, for example, to deliver volumes of approximately 0.15 mL peractivation.

Mechanisms of Action

DIPA-1-7 and DIPA-1-8 produce an anti-fatigue effect and provide reliefof heat stress and skin discomfort by evoking a sense of “dynamic cool”at sites of application. The sensation is not a steady cool, cold, oricy-cold sensation, but one of robust freshness, as if suddenly a fresh,cool breeze was blown on the skin (e.g., on the face). This effect,especially with DIPA-1−/is intense. The neurophysiological basis forthis sensation, possible receptor mechanisms, and the significance ofdynamic cooling for anti-fatigue, anti-heat stress, and anti-pruriticactions are further discussed herein.

Neurophysiology:

Small myelinated (Aδ) and unmyelinated fibers (C fibers) increaseafferent firing rate when skin temperature is lowered, for example,between 35° C. and 15° C. These neuronal signals that detect heatabstraction are transmitted to the central nervous system and generateconscious perception of coolness and cold. When skin temperature israised from 35° C. and 40° C., firing rates are increased in C fibersand these fibers signal warmth [Hutchinson et al. Quantitative analysisof orofacial thermoreceptive neurons in the superficial medullary dorsalhorn of the rat. J. Neurophysiol. 77, 3252-66, 1997]. The receptivemechanisms and “cable lines” for cool/cold and warm are separate anddistinct, but reciprocally inhibit each other in the brain and perhapsalso in the periphery. The sensory receptors are modality specific anddo not respond to mechanical stimulation. At the molecular level, thetarget binding sites for cooling agents are thought to be located on ionchannel receptors that depolarize in response to a drop in temperature.Heat abstraction decreases the threshold for discharge of the receptor,and the facilitated depolarization initiates the axonal responses thatcreate the neuronal signal.

The central response of these neurons has been recorded and studied fromrat superficial medullar dorsal horn that responds to innocuous thermalstimulation of the rat's face and tongue [Hutchinson et al., 1997]. Stepchanges of −Δ5° C. stimulated cells with both static firing rates andcells that had mainly dynamic properties [Davies et al. Sensoryprocessing in a thermal afferent pathway. J. Neurophysiol. 53: 429-434,1985]. Similar studies in cats and humans showed that step decreases intemperatures (dynamic changes), as low as Δ0.5° C./second, were readilydetectable by neurons and by psychophysical measurements [Davies et al.Facial sensitivity to rates of temperature change: neurophysiologicaland psychophysical evidence from cats and humans. J. Physiol. 344:161-175, 1983].

From a study of the spike patterns of neuronal discharge(impulses/second), it was clear that dynamic, and not static firingresponses to a change in temperature were the most powerful stimuli forgenerating coolness/cold sensations That is, the brain “sees”−Δ° C./tand not absolute ° C. Thus, a cooling agent that simulates −Δ° C./t onnerve discharge will produce “dynamic cooling”.

Relationship of Dynamic Cooling to Anti-Fatigue:

Dynamic cooling (versus static cooling/cold) is essential for ananti-fatigue effect. For example, if one is tired and driving a vehicle,turning on the air-conditioning and blasting the air onto the face willcounteract fatigue [dynamic cooling]. But just turning on the airconditioner to lower ambient temperature and being chilled inside thevehicle [static cooling] will not make much of a difference.

The topical therapy for enhanced performance and counteract fatiguedescribed herein circumvents the necessity for systemic drugs that actinvasively on brain chemistry. The benefits of the topical therapy areillustrated by the Case Studies described herein.

Receptor Mechanisms:

There is a general view that “TRP-” ion channel receptors (A1, M8, andV1 to 4) are the principal physiological elements for physiologicaltemperature detection. The TRPM8 receptor is the one that responds tosensory/cooling agents such as menthol and icilin [McKemy et al.Identification of a cold receptor reveals a general role for TRPchannels in thermosensation, Nature, 416, 52-58, 2002]. TRPM8 is aprotein with 1104-amino acid residues and has six transmembrane domains.Activation of this receptor by lowering ambient temperature results inopening of pores of transmembrane loop and non-specific cation entryinto the cell. Depolarization of TRPM8 receptors on sensory neurons maythen transmit signals primarily via Aδ (and some C) fibres.

While this concept for the role of TRPM8 in sensory physiology may bevalid for physical changes in temperature, the interpretation of thesensory effects of chemical agents such as menthol and icilin are morecomplex. Menthol not only stimulates TRPM8 in vitro, but also TRPV3, areceptor associated with warmth and glycinergic transmission [Macphersonet al. More than cool: promiscuous relationships of menthol and othersensory compounds. Mol Cell Neurosci 32:335-343, 2006: Sherkheli et al.,Supercooling agent icilin blocks a warmth-sensing ion channel TRPV3,Scientific World Journal, 2012: 982725, 0.2012:Cho et al. TRPA1-likechannels enhance glycinergic transmission in medullary dorsal hornneurons. J Neurochem 122:691-701, 2012]. Thus, menthol and icilin are“promiscuous” cooling agents and their specific sensory effects may notbe associated with any one particular receptor protein. A laboratoryreagent specific and selective for TRPM8 will be valuable for experimentand is not currently available.

The Applicant has screened a large database of cooling agents but,surprisingly, only DIPA-1-6 and DIPA-1-7 produced super-robust dynamiccooling on skin. DIPA-1-8 also produces strong cooling and its actionsare prolonged, but it does not quite have the super “wow” coolingeffects of DIPA-1-6 and DIPA-1-7. Other cooling agents are lessstimulating or have shorter durations of action and thus less suitablefor the uses contemplated herein. Thus, the DIPA compounds, by contrastto menthol and icilin, are ideal selective reagents to investigate TRPM8function.

It may be concluded that DIPA-1-7 and DIPA-1-8 bind to a site on avoltage-gated ion channel receptor located on a nerve ending that issensitive to a decrement in physical temperature. This event facilitatesneuronal depolarization to a cooling/cold signal, and an actionpotential is transmitted via A6 and C fibers towards the central nervoussystem. If the nerve ending is located on the facial skin, the signal isrecordable from dorsal surface of the trigeminal nucleus in thebrainstem. Further rostral transmission and integration of signals giverise to the perception of coolness/cold and its topographicalassociation with the site of stimulation.

When one examines the structure-activity relationships (SAR) of the DIPAcompounds, it is noted that when R₁═R₂=isopropyl and R₃=n-hexyl (C₆) orn-heptyl (C₇), then dynamic cooling is observed. Strong cooling of longduration is also obtained with R₃=n-octyl (C₈). However, whenR₁=R₂=sec-butyl and R₃=n-butyl to n-octyl (C₄ to C₈), dynamic cooling ispartially observed, but with much less intensity. As shown in thestudies described herein, this distinction between di-sec-butyl anddi-iso-propyl compounds is also seen in animal studies on shakingbehaviour, an indicator of cooling actions in the rat (because shakingis inhibited by heat).

Shaking behaviour is a rapid alternating contraction of the supinationand pronation muscles about the spinal axis, and can be readily observedand counted. Fur-coated and feathered animals—when wet and cold—shake,like a wet dog [Dickerson et al., Wet mammals shake at tuned frequenciesto dry. J. Royal Society, Interface 9, 3208-3218, 2012; Ortega-Jimenez,V. M. et al. Aerial shaking performance of wet Anna's hummingbirds. J.Royal Society, Interface 9, 1093-9, 2012; Wei, Pharmacological aspectsof shaking behavior produced by TRH, AG-3-5, and morphine withdrawal,Federation Proc. 40: 1491-1496, 1981].

“Wet-dog shaking” has been studied in detail in animals. Rats can shaketheir head, the upper torso, or the shaking can be sufficiently violentto affect the whole body and make the animal lose its balance. DIPA-1-7and DIPA-1-8 elicit the vigorous type of shaking. The purpose orsurvival value of shaking to fur-coated and feathered organisms is toremove water droplets trapped on or near the skin. Removal of the waterdroplets on or near the skin by shaking reduces the organism's need toexpend energy to remove the water by evaporation. The likely equivalentbehaviour to shaking in humans is shivering, a condition caused bygeneralized sensations of coolness/cold. Human subjects recovering fromthe deep hypothermia of anaesthesia manifest vigorous shaking; acondition called post-anaesthetic shivering.

Icilin(1-[2-hydroxy]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one)induces vigorous shaking in rats [Wei. Chemical stimulants of shakingbehavior. J. Pharmacy and Pharmacology 28: 722-724, 1976], Surprisingly,two potent p-menthane carboxamide cooling agents[(R)-2-[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-propionicacid ethyl ester, and[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-aceticacid isopropyl ester], which have EC₅₀ values similar to icilin at theTRPM8 receptor, do not evoke shaking (when injected at 50 mg/kg s.c. inmale rats and observed for 1 hour. Icilin activation at the TRPM8receptor is abrogated by a G805A mutation at the second to thirdtransmembrane loop, but the effects of menthol are not affected. It islikely that DIPA-1-6, DIPA-1-7, and DIPA-1-8 also have specific sites ofbinding and activation on the TRPM8 receptor which are not shared bymenthol or p-menthane carboxamides. Recent studies by Wei and Kuhn haveshown that DIPA-1-6 and DIPA-1-7 are still active on TRPM8 receptorswith the G805A mutation.

The studies described in Watson et al., 1978 [New compounds with thementhol cooling effect. J. Soc. Cosmet. Chem. 29: 185-200, 1978] showthat the presence of a polar oxygen moiety capable of acting as anacceptor of a hydrogen bond from the receptor is essential forbioactivity. A Hückel molecular orbital calculation (using MolecularModelling Pro v6.0.3, ChemSW Inc, Fairfield, Calif. 94534, USA) on theisopropyl analogs versus the sec-butyl analogues favours a slightlyhigher partial negative charge (0.007e) on the oxygen in the sec-butylentities, suggesting that the sec-butyl substituents facilitate a higheraffinity of the oxygen to the hydrogen binding site of the receptor.Thus it is possible that isopropyl, with a “looser” affinity canassociate and disassociate with the receptor more rapidly, favouring thegeneration of a dynamic onset and offset response of the receptor. Thisrapid interaction with the binding site will favour a more “dynamic” andintense stimulation of cooling and give rise to the phenomenon known asshaking.

Another possibility is that DIPA-1-7 has a dual action on TRPreceptors,so that it stimulates TRPM8 and, at higher concentrations, stimulatesTRPV1. The dual action will give a cold-hot synergy that might lead to amore dynamic cooling sensation.

TRPM8, TRPA1, and TRPV1 Receptor Assays:

The in vitro effects of test compounds were evaluated on cloned hTRPM8channel (encoded by the human TRPM8 gene, expressed in CHO cells) usinga Fluo-8 calcium kit and a Fluorescence Imaging Plate Reader(FLIPRTETRA™) instrument. To examine the specificity of the testcompounds, further tests were conducted on TRPV1 channels (human TRPV1gene expressed in HEK293 cells) and TRPA1 channels (human TRPA1 geneexpressed in CHO cells). The assays were conducted by ChanTestCorporation, 14656 Neo Parkway, Cleveland, Ohio 44128, USA.

Selection of Active Ingredient

Ideally, an active pharmaceutical ingredient (API) formulated fordelivery to the keratinized skin should be stable, non-toxic, andsufficiently long-acting and potent to activate the mechanisms thatresult in an anti-fatigue, anti-heat, or anti-nociceptive effect. TheAPI should be dissolved and evenly dispersed in a composition so thatduring manufacture the formulation maintains a constant concentration.The final product should meet standards of cleanliness and sterility.For purposes of formulation, the API can be a liquid at standardconditions of temperature and pressure (STP) and that is evenlydissolved in aqueous solutions at neutral pH and/or isotonicity.Sterility of the final product can be optimally achieved by usingpurified reagents and filtration through micropore filters, heating, orirradiation. Standard excipients, such as emulsifying agents, isotonicsaline, solvents, stabilizing agents, and preservatives, may be added tooptimize the formulations, but the important ingredients should bepreferably soluble in aqueous media such as purified water or a standarddermatological solvent.

For a given individual, the perceived sensation is a function of theparticular cooling agent, the dose, the vehicle used to carry thecooling agent, the method of topical delivery, and the nature of thetarget surfaces. The Applicant has screened a number of candidatecompounds, such as p-menthane carboxamides, on the facial skin (Wei.Sensory/cooling agents for skin discomfort. Journal Skin BarrierResearch 14: 5-12, 2012). The studies here identify DIPA-1-6, DIPA-1-7,and DIPA-1-8 as having the preferred desired properties of an idealanti-fatigue, anti-heat, and anti-nociceptive agent.

To summarize, the design concepts that lead to the selection ofDIPA-1-6, DIPA-1-7, and DIPA-1-8, as being suitable agents are:

-   -   The definition of a rationale for using a “dynamic cool”        sensation around the orbit and zygomatic to combat fatigue, and        describing the neurophysiology and mechanisms of this action.        This sensory effect in unusual and found in the DIPA compounds        but not found with structurally similar compounds.    -   Devising a delivery method for the API which avoids contacting        nociceptors on the cornea because that will result in sting/pain        and be aversive and not practical.    -   Finding an ideal compound (API) by experiment: DIPA-1-7 is water        soluble (a clear solution is obtained at up to 20 mg/mL in        distilled water), stable to heat, and exerts a “dynamic cool”        sensation for five to seven hours at an applied concentration of        1 to 10 mg/mL. Tachyphylaxis does not develop to repeat        applications.    -   Defining the receptor targets of these compounds in vitro, and        the selectivity of the chosen API.    -   Defining an in vitro isolated nerve preparation that shows an        anti-nociceptive action of DIPA-1-7, and showing that this        effect is abrogated on the nerve from a TRPM8 knockout mouse.    -   Defining an animal model (of “wet-dog shakes”) that will        illustrate the “dynamic cool” properties and allow further study        of mechanisms of action and the selective differentiation of        various analogs.    -   Conducting tests in human volunteers that show efficacy of the        DIPA compounds for reducing fatigue caused by chronic illness        and heat stress, and for increasing mental energy levels in the        normal person.    -   Conducting tests in human volunteers that show DIPA compounds,        especially DIPA-1-7, is effective for relieving sensory        discomfort of the skin, and thus may be used as an        anti-nociceptive or anti-pruritic agent, or as a diagnostic tool        for evaluating skin dysesthesias.        Applications

The DIPA compounds, when applied to keratinized skin, havesensory/cooling effects that mimic heat abstraction, but without achange in tissue temperatures. These compounds, especially DIPA-1-5,DIPA-1-6, DIPA-1-7, and DIPA-1-8, can also penetrate the skin barrier,reach nerve endings in the epidermis and dermis, and enter the systemiccirculation to exert cooling actions. These effects are obtained atsmall volumes, e.g., 0.1 to 0.5 mL, applied at a concentration of 1 to20 mg/mL, or 0.1 to 2% wt/vol. The onset of effect is rapid, less than 5minutes, and the sense of coolness is robust, refreshing, and strong.Compounds with similar bioactivity on the keratinized skin are notcurrently known or used in cosmetic or therapeutic applications. Anumber of new applications are possible with a molecule having suchunusual properties.

For conditions of increased sensations of perceived heat stress,DIPA-1-7 can also be used, for example, to increase athleticperformance, to counteract the discomfort of vasomotor symptoms, and tocounteract the discomfort of inflammation. In normothermic conditions,DIPA-1-7 applied to the facial skin may be used to enhance cognition andto alert and increase vigilance. DIPA-1-7 may also be used as adiagnostic agent for cold allodynia and hyperalgesia, as a laboratoryreagent to characterize TRPM8 functions, and as an adjunct in theformulations of numerous topical pharmaceuticals.

Heat Stress:

Thermal comfort is a technical term used by air-conditioning engineersto define “a state of mind in humans that expresses satisfaction withthe surrounding environment.” Maintaining thermal comfort for occupantsof buildings or other enclosures is one of the important goals ofarchitects and design engineers. For most people, the room temperaturefor thermal comfort is 25° C. (77° F.). Careful studies have documentedthat work performance and productivity (output/input) drop by 2% forevery increment of +1° C. above 25° C. up to 33° C. At officetemperatures of 28-30° C. (82-86° F.), there is increased sweating andcomplaints of headache, drowsiness and dullness, difficulty inconcentrating, and physical discomfort. For example, studies have shownthat increasing the indoor air temperature of a call center from 25° C.to 26° C. decreased the call response rate from 7.79 to 7.64 calls/hr, a1.9% loss [Tanabe et al., 2007]. An ambient temperature above 25° C. isthus a form of heat stress.

Energy consumption of buildings in China account for at leastone-quarter of the country's energy use, and sales of air-conditioningsystems in Brazil and India are on an exponential increase. This rise inenergy use has raised further concerns about global warming, but as mostpopulations now work indoors, energy costs must be balanced againstworker productivity. Basically, a worker's efficiency is better when heor she is kept cool. A method for combating mental lassitude from a hotenvironment, without incurring expenditures for energy, would haveeconomic benefits. In the Case Studies describe herein, it was foundthat application of DIPA-1-7 to the facial skin of a student preparingfor exams was useful in overcoming the discomforts of heat. Theapplication of a DIPA-1-7 0.5% gel onto the facial skin, especially onthe skin of the eyelids, and also on the skin of the neck, also providesrelief from heat.

Athletic Performance:

It is a natural desire of humans to want to perform better, eitherphysically or mentally. Recently, there has been an enthusiastic surgeof interest in the use of cryotherapy to improve athletic performance.Cryotherapy is defined as “ . . . the lowering of tissue temperature(locally or generally) by the withdrawal of heat from the body toachieve a therapeutic objective . . . ” External pre-cooling by heatabstraction, for example, by immersion in ice or by wearing a vestpacked with ice, can improve work endurance in a hot environment (see,e.g., Marino et al., 2002). An increase in physical work output of ˜5%can be shown for tasks of approximately 30 min [Grahn D A et al. Heatextraction through the palm of one hand improves aerobic exerciseendurance in a hot environment. J Appl Physiol 99:972-978, 2005] Heatexhaustion limits work and this occurs when core body temperatureapproaches 40° C. (104° F.). Pre-cooling (or internal cooling, forexample, by drinking an ice slurry) slows down the rate of heataccumulation.

Surprisingly, the improvement in athletic performance can be attained byincreasing the perception of coolness, without modifying coretemperature. Investigators showed that trained marathon runners wearinga commercial cooling collar (Black Ice LLC, Lakeland Tenn.) extended thetime to reach volitional exhaustion by 13.5% [Tyler et al. Cooling theneck region during exercise in heat. J. Athletic Training, 46, 61-68,2011]. Cooling of the neck dampened the perceived level of thermalstrain and delayed the point of voluntary termination of exercise.Participants tolerated a higher body temperature and heart rate whentheir neck regions were cooled.

In several studies with menthol, a chemical that produces sensations ofcoolness without a change in skin or core temperatures, it was noticedthat an increased perception of cooling, without a change in core bodytemperature, may also enhance better physical performance. This effectwas unexpected and attributed to menthol being a “positive” placebo[Gillis D J et al. The influence of menthol on thermoregulation andperception during exercise in warm, humid conditions. Eur J Appl Physiol2010; 110:609-618; Schlader et al. The independent roles of temperatureand thermal perception in the control of human thermoregulatorybehavior. Physiol Behav 103:217-224, 2011]. The surface of the face isdensely innervated with nerve endings that detect temperature. Theperipheral cool/cold detection system is associated with specific nervefiber discharges and precisely regulated so ±1° C. is easilydiscriminated. Over 92% of thermoceptive units on the face, especiallyaround the lips, respond to cooling and these neurons are tonicallyactive at room temperature (see, e.g., Hutchison et al., 1997).

It is likely than an agent such as DIPA-1-7 or DIPA-1-8, applied to theface, neck region, or chest will decrease heat discomfort and improveathletic performance. Vasomotor Symptoms (“Hot Flashes/Night Sweats” inPost-Menopausal Women):

Flushing (vasodilation) and sweating occur on the body when the brain'sthermoregulatory system perceives a need to lower body temperature.After menopause, at least one-third of women experience “hot flashes”(i.e., brief but repetitive episodes of feeling warm and flushed, anddaytime and nighttime sweating). Replacement estrogens may alleviatesymptoms but there are uncertainties if hormone replacement therapy(HRT) is safe. Sweating episodes that occur at night and in the earlymorning hours are especially inconvenient because the bed-sheets becomewet and it is burdensome to change the bed-sheets on a daily or frequentbasis. Episodes of “hot flashes/night sweats” can occur as often as onaverage 14 episodes per week. Aside from HRT, current alternativemethods of therapy, such as yoga, acupuncture, and phytoestrogens, havelimited if any effectiveness. Hot flashes are especially problematic forpost-menopausal women who have undergone treatment for breast cancer[approximately 500,000 individuals in the USA] because HRT cannot beprescribed.

The DIPA compounds are potent agents that can cross the skin barrier andbe absorbed into the bloodstream and exert systemic effects. Onepossible method of treating vasomotor symptoms may be to topicallyadminister DIPA-1-6 or DIPA-1-7 via a controlled-release patch. Thesystemic effects of the DIPA compound will then give rise to coolingsensations to counteract activation of central heat-loss mechanisms(vasodilatation and sweating). The patch may be applied at night to aconvenient location of the body, e.g., the skin above thesupraclavicular fossa or the skin above the sternomastoid muscle, andthe released DIPA compound would then inhibit the “night sweats.”Alternatively, the DIPA compound (e.g., DIPA-1-5, DIPA-1-6, or DIPA-1-7)can be applied locally to the skin as a gel, lotion, or cream.

Cognitive Enhancement:

Humans want to perform better, either physically or mentally. Chemicalsthat enhance performance belong to two categories: those that increasephysical capabilities, e.g., anabolic steroids or vitamins, and thosethat increase cognitive functions. Drugs that are “cognitive enhancers”(CEs) are also called nootropic drugs or neuroenhancers, and includesubstances such as caffeine, amphetamines, methylphenidate, nicotine,donepezil, and modafinil. The CEs are designed to enhance theindividual's capacity for tasks such as abstract thinking, attention,attitude, brainstorming, comprehension, recognition, creative thinking,critical thinking, increasing curiosity, executive functions, decisionmaking, eidetic memory, emotions and feelings, goals and goal setting,imagination, intelligence, introspection, lateral thinking, learning,memory, mental calculation, motivation, perception, personality andrecollection (recall).

Conscious perception of the visual world depends on the visual system tocapture image patterns on the retina and to deliver it to the brain forcognition and understanding. Cognitive functioning is the sum of memory,intelligence, creativity and attention. Human attention is furtherdivided into attentional tone (the state of vigilance) and selectiveattention (the ability to focus on and to execute a task without beingdistracted). The brain network for attention and its pharmacology hasbeen the subject of reviews (Lanni et al. Cognition enhancers betweentreating and doping the mind. Pharmacological Research 57: 196-213,2008]. The neurotransmitter mechanisms of some CEs have beeninvestigated. Drugs such as amphetamines and methylphenidate increasevigilance via catecholaminergic pathways and nicotine and donepizil mayaffect selective attention via cholinergic pathways. The visual systemis especially important to an organism's survival and it is estimated byneurophysiologists that at least 90% of the organism's brain activity isfocused on processing and interpreting visual sensory input.

Not all chemicals that affect brain/behavior enhance performance. Forexample, alcohol (ethanol) and cannabis are not cognitive enhancers. Adecrement in cognitive performance is called cognitive dysfunction (orimpairment) and can be manifested as fatigue, sleepiness, loss ofmemory, and inability to learn, to make decisions, to complete tasks, orto follow instructions. Cognitive dysfunction leads to decreased jobproductivity, transportation system accidents, inability to perform, anddaytime fatigue/sleepiness. Many conditions can lead to cognitivedysfunction and impairment including ageing, anxiety, depression,Alzheimer's disease, strokes, Parkinson's disease, narcolepsy, insomnia,disruption of circadian rhythms, obstructive sleep apnea, anddepression.

Drugs such as caffeine, amphetamines, methylphenidate, nicotine,donepezil, and modafinil have been used to enhance cognition and totreat fatigue. These compounds act invasively on brain chemistry. Thatis, the drugs require access of the active agent to the bloodstream, andfrom there to central nervous system, to act upon enzymes or receptors.Drugs such as amphetamines and nicotine have addiction liability. Evencaffeine can over-stimulate the nervous system and causes palpitations,irritability, tolerance, and dependence. There is a need for alternativemethods for cognitive enhancement and the treatment of tiredness.

Use of drugs such as CEs in the healthy, e.g., in the academic andbusiness environment, has been the subject of much recent debate[Talbot, 2009, “Brain gain. The underground world of “neuroenhancing”drugs”, The New Yorker, 27 Apr. 2009; H. Greely. Towards responsible useof cognitive-enhancing drugs by the healthy. Nature 456: 702-706,December 2008, 2008]. Here the proposed method of CE is achieved bytopical administration of an agent with a “dynamic cool” effect onto theexternal surface of facial skin and there is no direct invasion of brainchemistry.

It may be asked why cognitive functions should be enhanced by a DIPAcompound. If you ask a person from a cold climate (e.g., Norway, Russia,or Korea) if frigid air on the face will wake you up and think moreclearly, they will state that this in a known experience and an obviousfact. Frigid cold weather makes people think more clearly. The dynamiccool produced by DIPA-1-7 is a similar alerting event.

Without wishing to be bound by any particular theory, the Applicantproposes the following hypothesis as an explanation for this phenomenon.Approximately 200 million years ago certain organisms acquired theability to control metabolic heat production (endothermy) and tomaintain a constant internal body temperature (homeothermy). Thisevolutionary transition, from a “cold-blooded” to a “warm-blooded”physiology, enabled such species to better adapt and to survive in avariable environment. Although humans primarily evolved in a warmhabitat, migration has also exposed the species to cold. Coolness is thefirst signal to warn of the need for heat conservation and is apervasive and dominant neuronal signal for ensuring the organism'ssurvival because the metabolic machinery of the organism operatesefficiently at, and is dependent on, a constant temperature. In thepresence of cold, an organism thinks and plan for survival. Thiscircuitry is built into the brain, and serves as a template for arousaland enhancement of cognitive function.

In summary, the applicant has found that applying a dynamic coolingeffect to the facial skin, especially the periorbital area, creates anenhancement effect on cognition.

Illness-Related Fatigue:

Feeling tired, weary, and fatigued is a common experience and isconsidered an inconvenience that may be resolved by taking a nap,drinking a cup of coffee, or stopping whatever activity that brought iton. In many disorders, however, fatigue is a non-specific symptom withadverse consequences.

Fatigue, and its operational deficits, are recognized in this definitionby the Federal Aviation Administration [Salazar, 2013: Fatigue inaviation. Medical Facts for Pilots. Publication OK-07-193, prepared forFAA Civil Aerospace Medical Institute]:

“Fatigue is a condition characterized by increased discomfort withlessened capacity for work, reduced efficiency of accomplishment, lossof power or capacity for work, reduced efficiency of accomplishment,loss of power or capacity to respond to stimulation, and is usuallyaccompanied by a feeling of weariness and tiredness” (see, e.g.,Salazar, 2013).

Fatigue is recognized as an important problem for patients with advancedprogressive illness, especially cancer, as fatigue negatively affectsphysical, psychological, social and spiritual well-being, and quality oflife (QOL) [Minton et al. Drug therapy for the management ofcancer-related fatigue, Cochrane. Database. Syst. Rev, Jul. 7, 2010].This symptom is identified as a condition that requires management andresearch priority. For cancer-related fatigue: a consensus definition is“a common, persistent, and subjective sense of tiredness related tocancer or to treatment for cancer that interferes with usualfunctioning”.

Conditions that cause fatigue include: anxiety, boredom, depression,disruption of circadian rhythm or sleep, heavy physical exertion,excessive mental activity, treatment for cancer, chronic illness, andheat stress [see, e.g., Salazar, 2013; Stasi et al. Cancer-relatedfatigue: evolving concepts in evaluation and treatment. Cancer, 98,1786-801, 2003]. The definition used by the National Cancer Institutefor fatigue is a condition marked by extreme tiredness and inability tofunction due lack of energy. Fatigue may be acute or chronic (greaterthan 1 month duration), and, depending upon the accompanying symptoms,severity, and duration, it may be further classified as mild, moderate,or severe. Fatigue is a subjective sensation and its primary symptom isa complaint of tiredness. [National Cancer Institute: PDQ® Fatigue.Bethesda, Md.: National Cancer Institute. Date last modified Nov. 4,2011. Available at: http://cancergov].

Assessment instruments specific for fatigue have been developed such asthe Brief Fatigue Inventory, the Cancer Fatigue Scale, the FatigueAssessment Instrument, and the Multidimensional Fatigue Inventory. Theimportant questions asked of patients are: (1) Do you feel or have youever felt unusually tired? (2) If yes, can you indicate how tired youfeel on average on a scale from 0 to 10? (3) How much does thistiredness affect your daily life activities?

Related symptoms of fatigue are: complaints of generalized weakness orlimb heaviness, diminished concentration or attention, diminishedenergy, increased need to rest, decreased interest in engaging in usualactivities, insomnia or hypersomnia, experience of sleep asun-refreshing or non-restorative, difficulty in completing daily tasksattributed to feeling tired, perceived problems with short-term memory,and changes in emotional reactivity (e.g., sadness, frustration, orirritability). If five or more these symptoms are present every day ornearly every day during a 2-week period, then a diagnosis of medicalfatigue is made. Using these questionnaires it has been estimated thatfatigue is present at the time of diagnosis in approximately 50% ofcancer patients, and can increase to 60-96% of cancer patients duringtreatment.

In addition to cancer, other serious illnesses in which fatigue has beenexamined for interventions include chronic obstructive pulmonarydisease, motor neuron disease, cystic fibrosis, dementia, Parkinson'sdisease, human immunodeficiency virus/acquired immune deficiencysyndrome, and multiple sclerosis. Recognised potential causes of fatigueinclude anemia, dehydration, infection, malnutrition, pain, depression,disturbed sleep, anxiety, hypothyroidism, disease progression, andmuscle wasting and deconditioning. A feature of fatigue in thesepatients includes feeling tired without exertion and even after resting.Patients complain of a reduced capacity to carry out the normalactivities of daily living, slow physical recovery from tasks, anddiminished concentration.

Management of fatigue includes drugs such as antidepressants,analgesics, stimulants, anxiolytics and nutritional supplements.Non-drug methods include counseling on improved sleep practices,physical exercises, and relaxation techniques. Erythropoietin anddarbepoetin, drugs that stimulate red blood cell production, areeffective, but may decrease survival, and this adverse effect limitstheir use. In reviews of the literature, no drugs that work as centralnervous stimulants other than methylphenidate exhibit clearly identifiedbenefits to counter fatigue [Talbo, 2009.]. Fatigue is considered acondition that requires research priority because other adverse effectsof cancer treatment, namely, pain and nausea, are relativelywell-managed, but fatigue is not.

Topical application of a “dynamic cool” agent such as DIPA-1-7 ontofacial skin may have utility to counter-act fatigue, refresh, and toinvigorate.

Sensory Discomfort from Body Surfaces:

The potent “dynamic cool” sensations produced by DIPA-1-7 and DIPA-1-8were further evaluated for anti-itch (and other anti-nociceptive)effects on skin. As shown in the Case Studies described herein, a 20mg/mL solution, applied with a cotton-tipped applicator potently stoppeditching and discomfort caused by contact dermatitis in threeindividuals.

A topical medication that can relieve sensory discomfort has manyapplications including:

-   a) alleviation of irritation, itch and pain from various forms of    dermatitis (atopic, contact, and irritant);-   b) pain from burned, traumatized, diseased, anoxic, or irritated    skin (e.g., skin damaged by laser surgery, diabetic ulcers, sunburn,    radiation), and from procedures related to wound debridement and    wound healing;-   c) itch and discomfort from skin infections, insect bites, sunburn,    photodynamic treatment of skin (e.g., actinic keratoses, basal cell    carcinoma), lichen sclerosus;-   d) pruritus due to xerosis [especially dry skin itch of the    elderly], psoriasis, or seborrheic dermatitis;-   e) stomatitis, cheilitis, itching of the lips from cold sores or    gingivitis;-   f) pruritus ani, hemorrhoidal discomfort, pain from anal fissures,    pain or itch from anal fistulas, pain from hemorrhoidectomy,    perineal inflammation, anogenital skin inflammation and discomfort    due to various local causes such as incontinence, diaper rashes,    perineal inflammation;-   g) vulval pruritus and pain (e.g., from candidiasis or idiopathic,    such as vulva vestibulitis and vulvodynia), dyspareunia, anogenital    infections, including warts and sexually transmitted diseases,    fungal infections, viral infections of the skin (especially in    immunocompromised patients);-   h) nasal or upper airway discomfort from breathing obstruction,    e.g., congestion, rhinitis, asthma, bronchitis, emphysema and    chronic obstructive pulmonary diseases, dyspnea, sleep apnea and    snoring; and-   i) conjunctivitis, ocular surface irritation, pain from trauma and    corneal abrasions, and pain from eye surgery.

Of special interest, is the use of DIPA-1-7 and DIPA-1-8 for scalp itch,e.g., in seborrheic dermatitis and psoriasis; these end-points beingunmet medical needs. DIPA-1-7 may also be used to refresh the skinbefore application, or after removal of, cosmetics from the skin, toreduce the irritant effects of benzyoyl peroxide in the treatment ofacne, and to reduce sebum secretion and the appearance of an “oily”skin.

Diagnostic Agent for Allodynia:

Allodynia (pain due to a stimulus that does not usually provoke pain)and hyperalgesia (increased pain from a stimulus that usually provokespain) are prominent symptoms in patients with neuropathic pain [WasJensen et al., 2014). Allodynia and hyperalgesia in neuropathic pain:clinical manifestations and mechanisms. Lancet Neurology, 13: 924-935,2014]. Patients with neuropathic pain frequently suffer from painfulsensations induced by normally innocuous skin cooling, a conditioncalled cold allodynia [Wasner, G. et al. The effect of menthol on coldallodynia in patients with neuropathic pain. Pain medicine (Malden,Mass.) 9, 354-8, 2008]. Cold allodynia is frequent in diabetic patientswith pain, but a simple diagnostic tool for differentiating neuropathicpain from somatic pain is missing. An agent such as DIPA-1-7 applied tothe skin may be useful for such diagnosis and aid in the selection ofthe best method for therapy. A 40% menthol solution in alcohol has beenused as a challenge agent, but the results in the clinic have beenambiguous [Binder J. et al. Topical high-concentration (40%)menthol-somatosensory profile of a human surrogate pain model. J. Pain,12: 764-773, 2011].

Laboratory Reagent for the Study of TRPM8 Function

Menthol and icilin are traditional laboratory tools for the study ofTRPM8 in vitro and in vivo. The limitations of these molecules forcharacterizing TRPM8 functions are well-known: namely, the lack ofreceptor selectivity, and the difficulty of preparing formulations foradministration [Yin et al. Therapeutic opportunities for targeting coldpain pathways. Biochemical Pharmacology, 2014]. Solvents such absoluteethanol and dimethylsufloxide are used to dissolve menthol crystals andicilin, respectively. These solvents independently have pharmacologicalactions and may distort the effects of the active ingredient.Water-soluble laboratory reagents such as DIPA-1-5, DIPA-1-6, DIPA-1-7,and DIPA-1-8, will greatly facilitate experimental investigations ofTRPM8 pharmacology and physiology. These compounds have not been used inthe prior art.

Prevention of Post-Operative Hypothermia and Post-Anaesthetic Shivering:

Surgical patients with mild peri-operative hypothermia (33 to 36.4° C.)and post-anaesthetic shivering have a greater risk of adverse outcomes,including events such as decreased wound healing, increased bleeding,and morbid cardiac events [Buggy et al. Thermoregulation, mildperioperative hypo-thermia and post-anaesthetic shivering. Brit. J.Anaesth. 84: 615-628, 2000]. A study has suggested that a TRPM8 agonistsuch as menthol, by producing cold sensations, can elevate coretemperature [Tajno et al Cooling-sensitive TRPM8 is thermostat of skintemperature against cooling, PloS one 6: 2011]. An agent such asDIPA-1-7, by increasing sensitivity to cold, may be an useful as a drugtreatment against post-operative hypothermia. In the rat, injection ofDIPA-1-7 induces shaking, elevation of body temperatures, and ashortening of the duration of pentobarbital anesthesia, as measured byrecovery of the righting reflex. These pharmacological actions willcounter the depressive effects of anesthetics on body temperature.

Pharmaceutical Adjunct:

In pharmaceuticals or cosmeceuticals, the term “adjunct” is anadditional substance, treatment, or procedure used for increasing theefficacy or safety of the primary substance, treatment, or procedure orfor facilitating its performance. The DIPA compounds relieve sensorydiscomfort of the skin, have anti-nociceptive activity, and are activeat less than 1 minute after application. They are ideal adjuncts forpharmaceuticals and for cosmetics applied to the skin.

If the primary substance is an irritant, the adjunct may be used todecrease irritancy, and hence improve patient tolerance and compliance.For example, an adjunct such as DIPA-1-7 can be added an anti-acnepreparation containing benzoyl peroxide. Benzoyl peroxide, the primarysubstance, works as a skin peeling agent, increases cell turnover, andreduces P. acnes, but it is an irritant and can cause burning, swelling,and pain when applied to the skin. Similarly, imiquimod (Aldara®), whichis used as a primary substance to treat genital warts and skin cancercan cause blistering and pain, and an adjunct such as DIPA-1-7 orDIPA-1-8 may increase patient acceptance and compliance in the use ofthis drug.

An adjunct such as DIPA-1-7 may be used to increase the “apparent”efficacy of another primary ingredient, and thereby improve patientsatisfaction and adherence to a dosage schedule. For example, DIPA-1-7at about 0.5 to 2%, stops itching within minutes after application. Ifcombined with an anti-inflammatory steroid, the preparation may be moredesirable than the anti-inflammatory steroid alone, which takes longerto act. Anti-inflammatory steroids, such as hydrocortisone,triamcinolone, and clobetasol are used for sensory discomfort of theskin in disorders such as insect stings, contact dermatitis, atopiceczema, and psoriasis. The presence of DIPA-1-7 as an adjunct, inaddition to helping to stop the itch, may help reduce the dose or thefrequency of application of the primary ingredient, yet achieve anequivalent therapeutic effect. This adjunct benefit will be especiallybeneficial in the use of skin steroids because of the well-knownundesirable effects of collagen degradation, tissue thinning, andincreased susceptibility to infections. An adjunct that reduces dosageor promote greater efficacy of the primary ingredient has value. Otherprimary anti-pruritics are aluminum acetate, and strontium chloride orstrontium nitrate.

For skin disorders, compositions of the present discovery may also beused as adjuncts for procedures such as phototherapy, laser therapy,cryotherapy, or UV-therapy of the skin.

Pharmaceuticals that may be used, in combination or in sequence withadjunct DIPA compounds include anti-inflammatory steroidal agents,anti-inflammatory analgesic agents, antihistamines, sympathomimeticamine vasoconstrictors, local anesthetics, antibiotics, anti-acneagents, topical retinoids, drug for genital warts and skin cancer, drugsfor wrinkles and ageing skin, anti-hemorrhoidal agents, drugs for vulvaritch, skin moisturizers, and agents for keratolysis.

Examples of steroidal anti-inflammatory agents include hydrocortisone,clobetasol, clobetasol propionate, halobetasol, prednisolone,dexamethasone, triamcinolone acetonide, fluocinolone acetonide,fluocinonide, hydrocortisone acetate, prednisolone acetate,methylprednisolone, dexamethasone acetate, betamethasone, betamethasonevalerate, flumetasone, fluticasone, fluorometholone, beclomethasonedipropionate, etc.

Examples of anti-inflammatory analgesic agents include methylsalicylate, monoglycol salicylate, aspirin, indomethacin, diclofenac,ibuprofen, ketoprofen, naproxen, pranoproten, tenoproten, sulindac,tenclotenac, clidanac, flurbiprofen, fentiazac, bufexamac, piroxicam,pentazocine, etc.

Examples of antihistamines include diphenhydramine hydrochloride,diphenhydramine salicylate, diphenhydramine, chlorpheniramine maleate,promethazine hydrochloride, etc.

Examples of sympathomimetic amine vasoconstrictors include phenylephrinehydrochloride, oxymetazoline, naphazoline, and other imidazolinereceptor agonists used for nasal decongestant activity and for rednessand vasodilatation on the ocular surfaces.

Examples of local anesthetics include dibucaine hydrochloride,dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, pramoxinehydrochloride, tetracaine, tetracaine hydrochloride, oxyprocainehydrochloride, mepivacaine, piperocaine hydrochloride, etc.

Examples of skin moisturizer ingredients include the three categories ofhumectants, emollients and preservatives. Humectants, such as urea,glycerin and alpha hydroxy acids, help absorb moisture from the air andhold it in the skin. Emollients, such as lanolin, mineral oil andpetrolatum, help fill in spaces between skin cells, lubricating andsmoothing the skin. Preservatives help prevent bacteria growth inmoisturizers. Other ingredients that moisturizers may contain includevitamins, minerals, plant extracts and fragrances.

Examples of antibiotics include neomycin, erythromycin, and theanti-viral agent docosanol (Abreva®), and experimental agents such as N,N-dichloro-dimethyltaurine. Topical anti-acne agents include benzoylperoxide, resorcinol, resorcinol monoacetate, and salicylic acid. Otheragents to counter acne include topical retinoids such as adapalene andisotretinoin (Retin-A, Differen, and Tazorac). Examples of keratolyticsinclude such agents as, alpha-hydroxy acids, glycolic acid, andsalicylic acid.

The adjunct DIPA compound can be used for medications that are usefulfor human therapy as well as for veterinarian uses.

Study 1

Toxicity

Preliminary toxicological studies were conducted on DIPA 1-7. It was notmutagenic in the Ames test (Strains TA 98 and TA100, with and withoutliver activation) (tests conducted by Apredica, Watertown, Mass., USA).

DIPA-1-7, dissolved in 3% ethanol/97% 1,2-propanediol, or vehicle alone,was administered at 20 mg/kg perioral for 7 days (N=10 per group) tomale rats, and on the 8th day, the animals were euthanized with sodiumpentobarbital and the major organs (body, heart, liver, lungs, kidney,testes, brain) were removed and weighed. Heart tissues (ventricle andheart valves) and liver samples were stained with hematoxylin and eosinand the histology examined. There was no significant difference in bodyor organ weights between the two groups and the heart and liverhistology were normal.

Study 2

Tissue Temperature

The compounds of the present discovery simulate the sensations of heatabstraction, but do not alter tissue temperatures. The average foreheadskin temperature of subjects (N=5) was measured following application ofDIPA-1-7 (with a wipe at a concentration of 20 mg/mL in distilled water)to the forehead skin. The results are summarized in Table 5. Thesubjects noted the cooling effect of DIPA-1-7 on the skin which lastedfor 30-45 minutes; however, skin temperatures were not affected.

TABLE 5 Skin temperatures of human forehead after DIPA-1-7, 20 mg/mL.Temperature (° C.) Time Control DIPA-1-7 Before 37.3 37.4  0 minutes37.2 37.4 15 minutes 37.5 37.5 30 minutes 37.1 37.1 45 minutes 37.4 37.260 minutes 37.0 37.1Study 3Sensory Effects of Compounds on Facial Skin

When a test compound is applied to the skin, it is possible tocharacterize the resulting sensations. The quality of the sensationsproduced by individual compounds favours certain characteristics thatare distinct. The quality of the sensations evoked, their descriptors,and their proposed mechanism of action, are summarised in Table 6. Forany compound, there may be some overlap in activity, but usually onecompound occupies only one or two categories of sensations. For example,icilin is exclusively cool, with very little “cold”. DIPA-1-6 andDIPA-1-7 are exceptional in producing pleasant, robust “dynamic cool.”DIPA-1-8, 2-6, are 2-7 are strong cold-producing agents.

TABLE 6 Descriptor and proposed mechanisms of DIPA compounds on skin.Proposed Mechanisms on Type of Sensation Descriptor Sensory NeuronsInactive No effect — Cool, steady and pleasant Cool Balanced stimulationof static and dynamic Cold, constant, but limited Cold Higherstimulation of static by desensitization Dynamic cooling, robust DynamicHigher stimulation of dynamic cool/cold, strong refreshing cool Stingingcold, sometimes Icy cold Stimulation of dynamic and with irritationstatic, and also nociceptive sites

Even after the offset of the cooling/cold action, some of the compoundshave a “reservoir effect.” Experimentally, this is measured 1 hour afteroffset by placing a hot and then a cold towel over the site ofapplication and determining if the onset of cooling/cold returns for atleast 30 minutes. If this occurs, then there is a positive “reservoireffect”. The “reservoir effect” can also be provoked with air movement,but the conditions for air movement are difficult to standardize. The“reservoir effect” of DIPA-compounds in skin is most likely due toresidual drug that is reactivated to stimulate dynamic/static sensoryneurons.

In the studies described herein, the sensation of coolness/cold is ratedas 0, 1, 2, or 3 with: 0 as no change; 1 as slight coolness, or cold; 2as clear-cut signal of coolness or cold; and 3 as strong cooling orcold. The sensations are recorded at intervals of 5 to 15 minutes, untilat least two successive zeroes are obtained.

The onset of drug action is taken as the time to reach 2 units ofcoolness intensity.

The duration of sensory action is defined as the offset time minus theonset time. The offset of drug action is defined here as the time whencoolness intensity drops below 2, after previously surpassing 2 units.An inactive compound is defined as one that does not exceed 2 units ofcooling for 5 minutes or more after application. The offset endpoint issometimes unstable for compounds that act for two or more hours, becausethe coolness/cold sensation may fluctuate due to environmental variablessuch as sunlight, ventilation, activity, and the “reservoir effect.” Forexample, DIPA-1-8 and 2-8 are exceptionally long-acting on the skin.

The effects of test compounds on periorbital skin, malar (zygomatic)skin, and forehead skin were determined.

Compounds were tested on periorbital skin. Test compounds were appliedto the closed eyelids using cotton gauze (0.4 g, rectangular, 50 mm×60mm; from CS-being, Daisan Cotton, Japan). The test compounds were usedat a concentration of 1 mg/mL in distilled water. The duration of thesensory effect was measured with a stopwatch. The degree of “dynamiccool” was graded from 0 to +++, with intermediate steps of + and ++. Ananti-fatigue effect was present only if there was sufficient “dynamiccool.”

The results are summarized in Table 7.

TABLE 7 Sensory effects after application to periorbital skin. Sting onCarbon Sensory Anti- Duration Ocular Code R₃ atoms Quality fatigue (hr)Surface DIPA-1-5 5 11 dynamic + 0.5 No DIPA-1-6 6 12 dynamic ++ 3.8 YesDIPA-1-7 7 13 dynamic +++ 4.2 No DIPA-1-8 8 14 cool ++ 2.1 No DIPA-1-9 915 cool 0 3.0 No 2-4 4 12 cool 0 0.1 No 2-5 5 13 cool + 2.1 No 2-6 6 14cool ++ 6.2 Yes 2-7 7 15 cool + 1.2 Yes 2-8 8 16 cool + 1.3 No Compoundswere tested on zygomatic and forehead skin. Test compounds were appliedto the skin of the forehead and zygomatic using cotton gauze (0.4 g,rectangular, 50 mm × 60 mm; from CS-being, Daisan Cotton, Japan). Thetest compounds were used at a concentration of 20 mg/mL in distilledwater. The onset and duration of the sensory effect was measured with astopwatch. The degree of “dynamic cool” was graded from 0 to +++, withintermediate steps of + and ++. An anti-fatigue effect was present onlyif there was sufficient “dynamic cool.”

The results are summarized in the Table 8.

TABLE 8 Sensory effects after application to zygomatic and foreheadskin. Dura- Carbon Onset Sensory Anti- tion Reservoir Code R₃ atoms(min) Quality Fatigue (hr) Effect DIPA-1-5 5 11 ~1 dynamic 0 0.5 NoDIPA-1-6 6 12 ~1 dynamic ++ 1.3 Yes DIPA-1-7 7 13 ~1 dynamic- +++ 3.2Yes icy DIPA-1-8 8 14 ~1 cold-icy ++ 4.0 Yes DIPA-1-9 9 15 ~2 cool 0 2.0No 2-4 4 12 ~1 cool 0 0.3 No 2-5 5 13 ~1 cool 0 1.1 Yes 2-6 6 14 ~2cold + 1.5 Yes 2-7 7 15 ~2 cold + 2.4 Yes 2-8 8 16 5 cold 0 5.6 Yes

Each of 3-1 and 3-2 was tested and found to be inactive on periorbital,and zygomatic/forehead skin.

Notably, DIPA-1-7 selectively produced the unusual sensation of “dynamiccool” and also had anti-fatigue effects. From the data shown above, itcan be seen that, among these compounds, DIPA-1-7 evoked “dynamic cool”on both periorbital and zygomatic/forehead surface. Another compoundwith similar properties was DIPA-1-8, but this compound is was morecold/icy cold, although it had the desirable property of a longerduration of action on the zygomatic/forehead surface. The long durationof action of DIPA-1-7 and DIPA-1-8 on the skin adds value as ananti-fatigue agent, especially for the fatigue of chronic illness. Asshown in the case studies described below, a single application ofDIPA-1-7 is sufficient to counteract fatigue and heat stress for atleast three to four hours.

A special value of DIPA-1-9 is the comfortable cooling it provides andits long duration of action after periorbital application, and theabsence of any stinging. Thus, it has a special therapeutic niche forthe relief of ocular discomfort.

After further study, features in structure-activity relationships beganto reveal the attributes of DIPA-x compounds that predicted uniqueproperties by contrast to the 2-x series of compounds. The preferredembodiments of this application [for example, DIPA-1-7, DIPA-1-8,DIPA-1-9] are achiral, and exist as single defined molecular entities.There is a symmetrical branched isopropyl carbon chain adjacent to thephosphorus-oxygen group. Thus, there is a single molecular species forbioactivity, by contrast to the 2-x series which have four enantiomers.

Chirality is also an issue when a molecule is taken forward for drugdevelopment. The FDA and other regulatory agencies state that if a drugcandidate is a racemate, quantitative assays should be developed toexamine the pharmacokinetics of each enantiomer [US FDA, Development ofNew Stereoisomeric Drugs, Publication date May 1, 1992 and updated Dec.7, 2014]. It is also recommended that the pharmacological activity ofeach enantiomer should be evaluated in in vitro assays. Theseregulations compel the synthesis and testing of each enantiomer in aracemate. If a candidate compound is achiral, this extra work can beavoided. Technically, the separate synthesis of individual enantiomersis very challenging.

The preferred embodiments [DIPA-1-x series] not only differ in chiralityfrom other analogs in these series, but also have the advantages ofincreased water solubility over the chiral compounds because thephosphorus-oxygen atom is less masked by hydrocarbons. The preferredembodiments have a reduced likelihood to form emulsions or micelles, afact that is known to physical chemists who have studied thesecompounds.

The preferred embodiments described here are amphiphilic, that is,molecules have hydrophilic and lipophilic ends. Amphiphiles aggregate inan organized pattern in water to form micelles. For equivalent number ofcarbons, the DIPA-1-x series has more hydrophilic characteristics than2-x, as can be shown in miscibility studies. The geometry of the carbonsabout the phosphorus “head” of the molecule favors micelle formation forDIPA-1-x over 2-x molecules of equal molecular weight. Thus, thedifferences between the two set of compounds are inherent in thestructure. From the chemical structure, it can be seen that the presenceof the two di-sec-butyl groups on the phosphine oxide “pushes” the polarphosphorus group towards the center of the molecule, so the polar centeris less able to interact with water or to organize into micelles.

The sensory properties of the anti-fatigue effects of DIPA compounds andtheir duration of action may be correlated to the availability of thephosphorus oxide for hydrogen bonding. For the duration of action on theskin, increasing the number of carbons on R₃ increased the duration ofcooling, as might be predicted on the basis of lipophilicity, but theperiorbital effects indicate hydrophilicity is also important foranti-fatigue actions. In the section on “Receptor Mechanisms”, theimportance of a partial charge on the phosphinoyl oxygen for hydrogenbonding and an “on-off” or “rapid association-dissociation” foractivating dynamic cool are discussed. The results here for theselective attributes of DIPA-1-7, DIPA-1-8, and DIPA-1-9 are unexpected,surprising, and has practical applications for counter-acting fatigueand anti-nociception.

Study 4

Agonist Activity of Compounds on TRPM8

The in vitro effects of test compounds were evaluated on cloned hTRPM8channel (encoded by the human TRPM8 gene, expressed in CHO cells) usinga Fluo-8 calcium kit and a Fluorescence Imaging Plate Reader(FLIPR^(TETRA)™) instrument. The assays were conducted by ChanTestCorporation (14656 Neo Parkway, Cleveland, Ohio 44128, USA).

Test compounds and positive control solutions were prepared by dilutingstock solutions in a HEPES-buffered physiological saline (HBPS)solution. The test compound and control formulations were loaded inpolypropylene or glass-lined 384-well plates, and placed into the FLIPRinstrument (Molecular Devices Corporation, Union City, Calif., USA). Thetest compounds were evaluated at 4 or 8 concentrations with n=4replicates per determination. The positive control reference compoundwas L-menthol, a known TRPM8 agonist. The test cells were ChineseHamster Ovary (CHO) cells stably transfected with human TRPM8 cDNAs.

For FLIPR^(TETRA)™ assay, cells were plated in 384-well black wall, flatclear-bottom microtiter plates (Type: BD Biocoat Poly-D-Lysine MultiwellCell Culture Plate) at approximately 30,000 cells per well. Cells wereincubated at 37° C. overnight to reach a near confluent monolayerappropriate for use in a fluorescence assay. The test procedure was toremove the growth media and to add 40 μL of HBPS containing Fluo-8 for30 minutes at 37° C. 10 μL of test compound, vehicle, or controlsolutions in HBPS were added to each well and read for 4 minutes.

0.7

Concentration-response data were analyzed via the FLIPR Control softwarethat is supplied with the FLIPR System (MDS-AT) and fitted to a Hillequation of the following form:

${RESPONSE} = {{Base} + \frac{{Max} - {Base}}{1 + \left( \frac{xhalf}{x} \right)^{rate}}}$where: “Base” is the response at low concentrations of test compound;“Max” is the maximum response at high concentrations; “xhalf” is theEC₅₀, the concentration of test compound producing half-maximalactivation; and “rate” is the Hill coefficient. Nonlinear least squaresfits were made assuming a simple one-to-one binding model. The 95%Confidence Interval was obtained using the GraphPad Prism 6 software.

The results are summarized in Table 9.

TABLE 9 EC₅₀ and relative potency of compounds on TRPM8 . . . 95%Confidence Relative Code EC₅₀ μM Interval Potency Menthol 3.8 2.5 to 5.61.0 DIPA-1-5 5.6 4.4 to 7.2 0.7 DIPA-1-6 2.4 1.5 to 4.0 1.6 DIPA-1-7 0.70.5 to 1.0 5.4 DIPA-1-8 0.7 0.5 to 1.0 5.4 DIPA-1-9 0.9 0.4 to 2.5 4.02-4 14.5  7 to 29 0.3 2-5 1.7 1.0 to 2.9 2.2 2-6 0.8 0.5 to 1.3 4.7 2-71.1 0.6 to 2.3 3.4 2-8 1.3 0.7 to 2.3 2.9 3-1 24  8 to 76 0.2 3-2 4.2 1.6 to 10.8 0.9

Of the 12 compounds tested, all showed full efficacy on the TRPM8receptor, i.e., at higher tested concentrations there was ˜100%stimulation of calcium entry, and the data fitted a sigmoidaldose-response curve. The results for the “di-isopropyl” compounds ofthis invention are illustrated in FIG. 3.

FIG. 3 is a graph of response (Relative Fluorescence Units; % ofmaximum) as a function of the logarithm of the concentration of the testcompound (denoted agonist), expressed in μM, for each of DIPA-1-5(circles), DIPA-1-6 (squares), DIPA-1-7 (inverted triangle), DIPA-1-8(diamonds), or DIPA-1-9 (up-right triangle).

The EC₅₀ of the more potent compounds (DIPA-1-7, DIPA-1-8, DIPA-1-9,2-5, 2-6, 2-7, 2-8) fell within a narrow range with overlapping 95%Confidence Intervals. The potency of DIPA-1-7 and DIPA-1-8 are similarand significantly greater than the potencies of DIPA-1-5 and DIPA-1-6.By contrast the structural modifications of comparative compounds 3-1and 3-2 resulted in a significant loss of bioactivity

To examine the specificity of the test compounds, further studies wereconducted on TRPV1 channels (human TRPV1 gene expressed in HEK293 cells)and TRPA1 channels (human TRPA1 gene expressed in CHO cells). The testcells were Chinese Hamster Ovary (CHO) cells or Human Embyronic Kidney(HEK) 293 cells transfected with human TRPV1 or TRPA1 cDNAs. Thepositive control reference compound was capsaicin (a known TRPV1agonist) or mustard oil (a known TRPA1 agonist). DIPA-1-7 and DIPA-1-8did not exhibit any agonist on antagonist activity on TRPA1 channels atmaximum tested concentrations of 100 μM. A weak TRPV1 agonist activitywas found for DIPA-1-7, but this was not dose-dependent.

In bioactivity studies, potency was not correlated to the TRPM8 EC₅₀.For example, DIPA-1-5 and 1-6 are more potent in producing shakingbehavior than 1-7 and 1-8 [see Study 6]. Also, DIPA-1-7 is moreeffective at producing the sensation of “dynamic cool” on the skin andon the ocular surface. There were no distinguishing features in the EC₅₀data which enabled prediction of which compounds have potent “dynamiccool” properties. Thus, EC₅₀ values do not give information on thequality of the heat abstraction sensation, the duration of action, orthe accessibility of the molecule to tissue targets. The identificationof selective agents requires bioassays that more directly address thesequestions.

Study 5

Studies on Isolated Vagus Nerve: Direct Anti-Nociceptive Activity

To determine if DIPA-1-7 acted directly on sensory nerves, it was testedin an isolated nerve model developed at the Imperial College, London,U.K. [Birrell et al. TrpA1 agonists evoke coughing in guinea pig andhuman volunteers. Amer. J. respiratory and critical care medicine 180,1042-7, 2009; Patel, H. J. et al. Inhibition of guinea-pig and humansensory nerve activity and the cough reflex in guinea-pigs bycannabinoid (CB2) receptor activation. Brit. J. Pharmacol. 140, 261-8,2003]. In this in vitro assay, segments of the mouse vagus nerve areplaced on a platform and the electrical activity is recorded aftertopical application of capsaicin. Capsaicin is a known irritant thatelicits pain when it is applied to the skin and it will depolarize theisolated vagus. The ability of substances to inhibit thiscapsaicin-induced depolarization is measured.

Briefly, segments of vagus nerve, caudal to the nodose ganglion, wereremoved from mice with fine forceps and segments placed in oxygenatedKrebs solution and bubbled with 95% O₂/5% CO₂. The desheathed nervetrunk was mounted in a ‘grease-gap’ recording chamber and constantlysuperfused with Krebs solution with a flow rate of approximately 2mL/min, and the electrical activity of the nerve monitored withelectrodes. The temperature of the perfusate was kept constant at 37° C.by a water bath. Nerve depolarizations were induced by superfusion ofthe nerve with capsaicin (1 μM). After two reproducible depolarizationresponses to capsaicin, DIPA-1-7 was applied at 1 mg/mL (4 μM) for 10minutes in the perfusate followed by capsaicin. The nerves were thenwashed with Krebs until the responses had returned to baseline andchallenged again with capsaicin. The results and tracings obtained innormal and TRPM8 knockout mouse are shown in FIG. 4.

FIG. 4 shows chart traces that illustrate, in the first trace (“WildType”), the inhibition of capsaicin-induced depolarization of theisolated mouse vagus by DIPA-1-7, superfused at a concentration of 1mg/mL, and, in the second trace (“TRPM8 KO”), the significant absence ofinhibition in the isolated TRPM8 KO (knockout) mouse vagus by DIPA-1-7,superfused at a concentration of 1 mg/mL.

In the tracings shown in the FIG. 4, the first two peaks show thedepolarization response of the mouse vagus to capsaicin (“Caps”). AfterDIPA-1-7 is applied (1 mg/mL), the response is suppressed in the normalmouse vagus (“Wild Type”), but not in the TRPM8 knock-out (“TRPM8 KO”)mouse vagus.

The percent inhibition of capsaicin-induced depolarization of theisolated normal mouse vagus caused by DIPA-1-7 was about 75%; thepercent inhibition of capsaicin-induced depolarization of the isolatedTRPM8 knock-out mouse vagus caused by DIPA-1-7 was about 20%.

This experiment clearly demonstrates a direct pharmacological action ofthe DIPA-1-7 on the sensory nerve, which is a surprising and unexpectedresult. Furthermore, the diminished response in the TRPM8 KO mouseindicated that the receptor target was TRPM8. These results providestrong evidence that DIPA-1-7 can be used as an anti-nociceptive agentand the target receptor is TRPM8.

Capsaicin is a TRPV1 agonist and the search for an effective TRPV1antagonist has been the super-intense quest of many pharmaceuticalcompanies for the past ten or more year. Here, it is shown that DIPA-1-7is an effective “physiological” antagonist of TRPV1 at lowconcentrations. DIPA-1-7, by itself, did not evoke depolarization,indicating that it is free of agonist activity at this “pain” receptor.These results strongly indicate the usefulness of DIPA-1-7 as ananti-nociceptive agent.

Study 6

Activity in Laboratory Rat: Perioral, Topical and Intravenous Delivery

Fur-coated and feathered animals—when wet and cold—shake, like a wet dog(see, e.g., Dickerson et al., 2012; Ortega-Jimenez et al., 2012; Wei,1981). These shakes are rapid alternating contractions of the supinationand pronation muscles about the spinal axis, and can be readily observedand counted. “Wet-dog shaking” has been studied in detail in animals andthis behaviour is interpreted to have survival value because shaking, byremoving the water off t skin, reduces the need to expend evaporativeenergy to remove wetness. The triggering sensation for shaking is thushaving water trapped in between hair follicles or feathers. Humans havelittle hair on skin and do not shake. The likely equivalent behaviour toshaking in humans is shivering, a condition caused by generalizedsensations of coolness/cold and wetness.

Drug-induced shaking in animals has been reviewed (see, e.g., Wei,1981). Under the right conditions, drug-induced shaking can be observedin the pentobarbital-anesthetized rat, enhanced by hypothermia and cold,and inhibited by elevating body temperature.

In experiments conducted here, test compounds were evaluated for“wet-dog shaking” as a model of dynamic cooling. Using a standardizedprocedure, test compounds were compared in their ability to stimulatethe shaking response by perioral administration, by topical delivery tothe abdominal skin, and by intravenous administration through acannulated femoral vein.

Perioral.

Test compounds were dissolved in saline and administered by oral gavageto pentobarbital-anesthetized male albino rats at 20 mg/kg at a volumeof 0.1 mL/100 g body weight [N=3 to 4 rats per compound]. Shaking wascounted over a 40 min period and recorded at 10-min intervals.

Three of the four “di-isopropyl” compounds caused vigorous shaking. The“di-secbutyl” compounds were relatively inactive, except 2-5 whichelicited an average of 4 shakes in the 40 min observation period. Bycontrast, DIPA-1-5, DIPA-1-6, and DIPA-1-7 produced an average shakingfrequency of 86, 56, and 36 shakes, respectively. The strong activity ofDIPA-1-5 was unusual. Applied to the skin, DIPA-1-5 has a refreshing“dynamic cool”, but the duration of action of only about 30 min wassignificantly less than that for DIPA-1-6 and DIPA-1-7. The shorterduration of action of DIPA-1-5 limits its practical utility. It ispossible that its smaller molecular size facilitates absorption andallows greater access to systemic receptors, and therefore more shaking.

The relationship of the shake response to temperature sensation wasfurther studied [in pentobarbital-anesthetized rats]. After injection ofthe sodium pentobarbital anesthetic, rectal temperature drops, andreaches approximately 35° C. in about 10 min. This hypothermia can bereversed by placing the animal on a heated surface and body temperaturemaintained at 38° C. DIPA-1-7 20 mg/kg perioral elicited 36±5 shakes(N=6) in the anesthetized rat, but in the heated animals, the shakingfrequency was significantly reduced to 5±2 shakes (N=6) [P<0.001]. Thereduction of shaking frequency by ⅔ under heat indicated that the shakeresponse was linked to cold sensations and shivering.

Topical.

Shaking is an excellent indicator of in vivo effect. Methods weredeveloped to determine if shaking was seen after topical application ofDIPA compounds. The abdominal skin of the pentobarbital-anesthetized ratwas shaved and 20 μL of the pure unadulterated DIPA chemical was appliedwith a micropipette on a ˜1 cm diameter circle of skin, enclosed with aring of cream [Baby cream “Nevskaya kosmetika Detskyi” NevskayaKosmetika Inc., Saint-Petrsburg 192029], as shown in FIG. 4. The numberof shakes was counted for 1 hr after application.

FIG. 5 shows the method for measuring the transdermal activity ofDIPA-compounds applied 20 μL with a micropipette to the center of acircle enclosed by cream on the abdominal skin of an anesthetized rat.Shaking frequency was counted for 1 hr after topical application. Thedata and results for topical for perioral responses are summarized inthe Table 10. The data are further plotted graphically in FIG. 6, toshow the lack of correlation of TRPM8 potency to in vivo bioactivity bythe topical route of administration.

Surprisingly, vigorous shaking was evoked with inventive embodimentsDIPA-1-5, DIPA-1-6, and DIPA-1-7. Only a weak response was seen withDIPA-1-8, and the comparative di-sec-butyl analogs, 2-5, 2-6, and 2-7,were inactive. The shaking induced by DIPA-1-7 was dose-dependent.Topical application of 5 μl, 10 μl, 20 μl, or 50 μl of DIPA-1-7 elicitedan average of 25±3, 53±6, 79±8 and 118±12 shakes, respectively, in 1 hr.

The data in Table 10 and FIG. 6 provide the STRONGEST evidence for thenovelty and exceptional properties for the compounds of this discovery.It is clear that these compounds penetrate biological membranes andrapidly evoke responses; events that are not seen with the comparativedi-sec-butyl analogs. By these routes, the bioactivity is not correlatedto the potency measurement [EC₅₀] on the TRPM8 receptor but isdetermined the more efficient penetration. This is the first time thatshaking responses of such magnitude have been shown after topical[dermal] application of a chemical.

Shaking was seen if DIPA-1-7 was diluted 50-50 with either water orsaline (at the 10 μl dose), but it was completely inhibited if 50%(R)-1,2-propanediol was added to the DIPA-1-7 (at the 10 μl dose) as adiluent. This surprising result shows that DIPA-1-7 penetrates the skinin aqueous solution. This facile permeability of DIPA-1-7 is reminiscentof menthol, and suggests DIPA-1-7 is easily delivered into the dermis bytopical application. From this observation, one can surmise thatDIPA-1-7 may be used to penetrate thick keratotic skin lesions, forexample in psoriasis or in contact dermatits of the hands, to alleviateitch and pain. The adjustment of DIPA-1-7 concentrations in polyhydricsolvent such as 1,2-propanediol can be used to control the degree ofabsorption of DIPA-1-7, an art well-known to formulation experts.

TABLE 10 Shaking frequency after perioral [per 20 mg/kg body weight] ortopical delivery of 20 μl test compounds [per animal] to theanesthetized rat. Mol # Code Wt Cs Sensation Perioral Topical xMentholDIPA-1-5 204 11 dynamic cool 86 ± 7 138 ± 15 0.7 DIPA-1-6 218 12 dynamiccool 56 ± 5 69 ± 8 1.6 DIPA-1-7 232 13 dynamic cool 36 ± 4 79 ± 8 5.4DIPA-1-8 246 14 cool 0  7 ± 2 5.4 DIPA-1-9 260 15 mild cool 0 0 4.0 2-4218 12 cool 0 0 0.3 2-5 232 13 cool  4 ± 1 0 2.2 2-6 246 14 cool 0 0 4.72-7 260 15 cool 0 0 3.4 2-8 274 16 cool 0 0 2.9

The surprising potency of DIPA-1-5 and DIPA-1-6 was unexpected. Thesemolecules work for a shorter time on skin cooling than DIPA1-7. Thesesmaller may penetrate faster through the skin barrier and go into thesystemic circulation. However, the value of this fast action isuncertain. In most contemplated topical applications of this discovery,the preference is for the drug action to remain localized and notsystemic.

Intravenous.

When the relative activities of the analogs for producing shaking werecompared to the EC₅₀ for TRPM8 activation [as measured by the xMentholpotency] it can be seen that the two variables are not correlated. Forexample, 2-6 is 4.7× menthol, but does not produce shaking by perioralor topical administration. Yet DIPA1-7, which 5.7× menthol, producesvigorous shaking by these routes. The lack of quantitative correlationis perplexing, as it is clear that the cooling properties are linked toTRPM8 activation. To clarify the discrepancy, DIPA1-7 and 2-6 werecompared by the intravenous [i.v.] route of administration, a deliveryroute which is not influenced by membrane barriers.

Male rats weighing ˜220-240 g were anesthetized with sodiumpentobarbital, 55 mg/kg intraperitoneal, and after the loss of therighting reflex, animals were place on a heated table and bodytemperature maintained at 37 to 38° C. The femoral vein was cannulatedwith PE-20 tubing connected to a 1 mL syringe. Stock solutions ofDIPA-1-7 and 2-6 were prepared in normal saline at 10 mg/mL and furtherdiluted to 1 mg/mL on the day of the experiment and injected at 0.1mL/100 g body weight to give a dose of 1 mg/kg i.v. Animals were pairedwith N=6 per group. Shaking frequency was counted for 30 min after i.v.delivery and the results compared with the Student's t-test. Two trialswere conducted per animal with a 1o to 15 min interval between doses.

The shaking frequency after DIPA-1-7 and 2-6 in this experiment areshown in FIG. 6. Shaking was observed immediately after i.v. injectionand at least 78% of the total shakes occurred in the first 5 min afterinjection. The response in the second trial was at least as robust inthe first trial, showing the lack of desensitization. For 2-6, theresponse in the second trial was more vigorous than in the firstinjection; indicating possible cumulative effects or a lightening ofanesthesia. The robust activity of 2-6 i.v. is in sharp contrast to theresults seen after perioral or topical delivery. These results providestrong objective laboratory evidence that the DIPA compounds of Formula1 are qualitatively different from the corresponding di-sec-butylcompounds: producing shaking by all three routes of administration,whereas the di-sec-butyl compound is active only by i.v. delivery.

In practice, topical penetration a the key factor for a successfulproduct if the receptor target is beneath the stratum corneum. Thus, thediispropyl analogs are better and different from the di-sec-butylanalogs. Another advantage, mentioned on page 15, is the absence of achiral center in the diisopropyl molecule.

Study 7

Effects on Topical Sites on the Cranium

DIPA-1-7, the most potent compound for dynamic cooling, was tested atother topical sites on the cranium. A 20 mg/mL solution was applied,using a cotton wipe, onto the skin above the buccal cheek, theparotid-masseteric cheek, temple, and the skin above the periauricularregion, and the posterior mandible using the appropriate craniometricpoints (pterion, coronion, condylion, and gonion, respectively) aslandmarks. Surprisingly, at all of these sites, other than the buccalcheek, little cooling, if any, was observed. Mild cooling was observedon the buccal cheek for approximately 30 minutes, but this effect mayhave been due to the spread of the solution onto the receptive field ofthe infraorbital nerve. Thus, the action on orbit and zygomatic/foreheadskin is selective and identifies the important delivery targets on theskin of the head.

The head is known to be a site where cooling helps relieve heatdiscomfort. In a study described in Nakamura et al. [2012], eleven malesubjects were exposed to mild heat. Subjects, clothed in only shortpants, entered a climatic chamber maintained at 32.5±0.5° C. with arelative humidity of 50%. About 1.5 hours after entry into the chamber,a local cooling protocol was initiated with water-perfused stimulatorsplaced on the head, chest, abdomen, or thigh. Cooling of the face andthigh was felt by the subjects to be more effective than cooling of thechest and abdomen in reducing the heat discomfort.

In a study described by Essick et al. [Site-dependent andsubject-related variations in perioral thermal sensitivity.Somatosensory & motor research 21, 159-75, 2004] the thresholds fordetection of cooling and cold pain on various sites of the face, ventralforearm, and scalp was determined for 34 young adults. The mostsensitive sites were on the vermilion which could detect a temperaturechange of about 0.5° C., followed by areas around the mouth (upper andlower hairy lip, mouth corner) and lateral chin. The mid-cheek andperiauricular skin were less sensitive (able to detect a temperaturechange of about 2° C.), and the forearm and scalp were least sensitive(able to detect a temperature change of about 3° C.). The sensitivitiesof the orbital, zygomatic and forehead skin were not tested.

Use of DIPA-1-7 on the orbital and zygomatic/forehead skin, for example,in an office environment or in heat stress, may be inconvenient if thesubjects uses cosmetic make-up at these sites. Surprisingly, it wasfound that DIPA-1-7, at 20 mg/mL, can produce a dynamic cooling effectwhen applied on the scalp, especially near the hairline. This effect issufficient to counter fatigue caused by heat. Likewise, rubbing DIPA-1-7on the skin in the centre of the chest, above the sternum, cancounteract the discomforts of heat. At these application sites,cosmetics are not affected, yet an invigorating coolness, thatcounteracts the debilitating effect of heat, is achieved.

The ability of DIPA-1-7 to cause cooling of the scalp and hairline isalso important for treating itch at these sites in conditions such aspsoriasis, dandruff, and seborrheic dermatitis. In September 2016, DongWha Pharmaceuticals, the oldest drug company in S. Korea introduced aproduct called Intrinsic IB [itch block} for the treatment of skindysesthesias caused, for example, by atopic dermatitis or psoriasis.

Study 8

Identification of TRPM8 Targets on the Eyelid Margins

TRPM8 is the principal receptor protein of cold-sensitive nerve fibersassociated with the detection of cooling sensations on body surfacessuch as the skin. TRPM8 immunoreactivity has been detected on the rodentcornea [Alamri et al. Transient receptor potential cation channelsubfamily V member 1 expressing corneal sensory neurons can besubdivided into can be subdivided into at least three subpopulations.Front Neuroanat. 2015: 9:71]. Activation of corneal TRPM8 causeunpleasant sensations of irritation [Kovacs et al. Abnormal activity ofcorneal cold thermoreceptors underlies the unpleasant sensations in dryeye disease, Pain 157, 399-417 (2016)]. Here, we used mice engineered toexpress the enhanced green fluorescent protein (EGFPf) from the TRPM8locus (TRPM8EGFPf). Unexpectedly, a dense TRPM8 innervation in theeyelid margins was found, especially at the base of the eyelash hairshafts, but hardly any TRPM8 fibers were found on the conjunctiva.Whorls of TRPM8 were also found at the ducts of the Meibomian gland, andpictures of the nerve network closely resembled the histologicalphotomicrographs shown in Montagna and Ford [Histology and cytochemistryof human skin: the eyelid. Arch. Derm. 100: 328-335, 1969]. This is thefirst time that TRPM8 nerve endings in the eyelid skin have been seen.

The sensory fibers innervating the upper eyelid and cornea are locatedin the V1 ophthalmic branch of the trigeminal nerve. We reasoned thatTRPM8 signals from the ocular margins may send cooling signals to thebrain and be perceived as pleasant and soothing by the supra and infraorbital nerves. By contrast, the corneal nerve endings will send painsignals via the ciliary nerve. Any stimulus from the corneal surfacewill be perceived as noxious.

TRPM8 receptors were identified on the eyelid skin by the presence ofgreen fluorescence in sensory nerve endings as visualized under themicroscope. Dr. Yu-Qing Cao, Pain Center of Washington University in St.Louis, School of Medicine, Missouri, provided the gift of Trpm8+/−:eFGPmice, and the studies were conducted by Li FengXian MD, PhD, and Prof.Liu Qin of the Department of Anesthesiology and Center for the Study ofItch, Washington University, School of Medicine, St. Louis, Mo., USA.The photomicrographs of the TRPM8 green fluorescence in the eyelids weresubmitted for publication. Dense innervation was observed in the eyelidskin, especially at the base of hair shafts of the lashes. Very fewfibers were found on the conjunctiva. Thus, the receptor targets for thewipe/pad/applicator method were identified. This is the first time thatthe TRPM8 nerve endings in the eyelid skin have been visualized. Thecornea was also innervated with nerve fibers, but if this target is incontact with the drug agent via eye drops there is stinging and pain.The sensory fibers innervating the upper eyelid and cornea are locatedin the V1 ophthalmic branch of the trigeminal nerve. We speculated thatTRPM8 signals from the ocular margins may be perceived as coolingsignals for the entire eye surface and gate nociceptive input from thecornea.

Trpm8EGFPf/+transgenic mice were anesthetized with xylazine (3 mg/kg)and ketamine (15 mg/kg) mixture for dye injection and perfusion. Micewere perfused with 4% paraformaldehyde in PBS (pH 7.2, 4° C.), followedby PBS (pH 7.2, room temperature). Tissues were sectioned at 12 μm ontoslides for study. Whole-mounts of upper eyelid skin were collected fromTrpm8EGFPf/+ mouse and post-fixed with 4% paraformaldehyde on ice for 2hr after CO2 euthanasia, then washed with PBS for 3 times beforeimmunostaining. Slides and upper eyelid skin were washed with PBS in0.2% Triton X-100 (PBST) for 3 times and blocked with 10% donkey serumin phosphate buffered saline with Tween 20 for 2 hr, then were incubatedin chicken anti-GFP (GFP-1020; Ayes Lab; 1:1000) solution at 4° C. for24 hr. Donkey anti-chicken IgG (114050, FITC conjugated; JacksonImmunoResearch; 1:1000) was incubated for 2 hr at room temperature after3 times washes with PBST. Sections and whole mount of eyelid skin,cornea, and dissected conjunctiva were washed with PBS and mounted withFluoromount-G (Southern Biotech). Images were taken and analyzed usingNikon fluorescence microscope with a CoolSnap HQ2 CCD camera(Photometrics, Tucson, Ariz.).

Case Studies

Case studies are described below which demonstrate the use of DIPA-1-7:(a) to enhance cognition, decrease mental lassitude and fatigue, and toenergize performance; (b) to counteract tiredness and fatigue fromchronic illness; (c) to counteract the fatigue and/or discomfort fromheat stress; (d) to counteract skin itch and pain, and (e) to reduce theseverity of “night sweats”.

In these studies, subjects were given dosages units containing 1.5 to1.75 mL of DIPA-1-7 stored in 2.0 mL microcentrifuge tubes (NovaBiostorage Plus, Canonsburg, Pa. 15317) and cotton gauze (0.4 g,rectangular, 50 mm×60 mml; from CS-being, Daisan Cotton, Japan). TheDIPA-1-7 was provided as a solution in distilled water or 2% ethanol-98%distilled water, at a DIPA-1-7 concentration of 1 mg/mL or 5 mg/mL. Thesubjects were given instructions on how to place the solution on thegauze and how to wipe the wet gauze over the skin surfaces with the eyesclosed: 5 mg/mL for the orbital and zygomatic/forehead skin, away fromthe palpebral sulcus, and 1 mg/mL if the primary site was theperiorbital skin. Approximately 0.35 mL and 0.15 mL are delivered bythese methods of application, respectively.

For some test (comparative) compounds (e.g., 2-6 and 2-7), residues thatremain on orbital skin can enter the ocular surface and cause stingingand discomfort when a subject sweats or takes a shower. This problem wasminimal with inventive embodiments DIPA-1-7 and DIPA-1-8. Subjects wereinstructed to rinse with water or a wet towel any surfaces that becomeirritable; however, irritation and discomfort was rarely seen withDIPA-1-7 or DIPA-1-8 at these concentrations.

Case Study 1

A 65-year old male is an avid snooker player and likes to frequent thesnooker parlours of London and Hong Kong. He plays for small wagers withhis friends, but with advancing age his game has deteriorated and he canonly play about eight frames in one day. He uses ice-cold towels on hisface and prescription glasses to help him during games, but feels thatit is the lack of concentration and the planning of sequences of shotsthat hinders his game and prevents him from completing “breaks” (acontinuous accumulation of points in a “run”). He volunteered to trywipes containing DIPA-1-7. There was a remarkable transformation in hisgame. He moved faster from shot to shot and the planning and executionwas crisp. The number of frames per session increased as well as hisfrequency of play. He had his longest career break of 80 points and wasecstatic. He continues to use the wipes as an aid to his snooker game.He also noted that enhancement of his cognitive facilities could berenewed and invigorated by applying the ice cold towel to his face (anexample of the “reservoir effect”). He noted, however, it was importantto avoid excessive entry of the DIPA-1-7 onto his ocular surface becausethat sometimes caused irritation, especially if the use was toofrequent. With practice, he noted that cognitive enhancement of his gamecould be regulated and controlled by optimizing the delivery procedures.

A 70-year old retired architect likes to play penny poker once or twicea week with his buddies. He volunteered to try wipes containing 5 mg/mLof DIPA-1-7 to see if it would improve his poker skills. He did this atfirst without telling his friends. He immediately noticed afterapplication of the wipe that he was more awake than the other players.He could remember the cards that were discarded, could calculate andremember the odds of various hands (e.g., likelihood of drawingsuccessfully to a four-card two-way straight or a four-card flush), butmost importantly, he could also sense if his opponent had a strong orweak hand, and if they were bluffing. He felt energized, moreadventurous, and willing to take risks by bluffing himself. He madedecisions quickly and with more confidence. He felt that his game wasmore insightful and improved. He felt guilty about having an unfairadvantage over his friends and encouraged several of the other playersto try the wipes. All noticed the invigorating dynamic cool sensationsbut they were less sure if their poker skills were improved.

A 68-year old pharmacologist spends his time in research and in thedesign and management of clinical trials. He owns his consulting firmwith eight employees, and spends at least 8 to 12 hours per day in frontof a computer monitor. He has in his working space an espresso machine,and boxes of cigarettes and cigars. He uses coffee and tobacco tosharpen his thinking. He agreed to apply the wipes containing DIPA-1-7at 1 mg/mL (periorbital only) and 5 mg/mL (periorbital andzygomatic/forehead) and noted that his tiredness went away for at least6 to 8 hours and that he was able to concentrate and think more clearly.He said the wipes were superior to both coffee and tobacco in improvinghis concentration. He now also uses the wipes for work and beforebusiness and scientific meetings to enhance his social performance andmental acuity, and to reduce fatigue.

A 72-year old retired policeman decided to return to work as a securityguard because he needed the funds to support his grand-daughter'scollege costs. He worked from noon to 8:30 pm and complained ofweariness and fatigue which affected his activities. He said he was sotired that he could not stay awake for televised football games, eventhough he was an avid fan. He volunteered to try the wipes containingDIPA-1-7 and said they definitely made him more vigilant, especiallywhen driving home from work. He said that turning on the car'sair-conditioning so that the cool air vent was aimed at his face,together with menthol mints and the wipes, kept him alert, and that heno was no longer a threat on the roads. He had an 18.5 inch (47 cm)neckline and snored heavily at night, but polysomnography did not revealsleep apnea episodes. He felt that by using the wipes on his orbit somecoolness drained down onto his nasal membranes (via the nasolachrymalduct), and that this cooling sensation in his nose allowed him tobreathe more freely and to sleep better at night. Currently, he isexercising more and trying to reduce food intake, in order to controlhis fatigue.

Several individuals also tried the wipes containing DIPA-1-6, DIPA-1-8,2-6 and 2-7, and also found these compounds to be effective forenhancing performance and thinking, but the effects were consideredsomewhat less dramatic, or with some residual sting. Of these analogs,DIPA-1-8 was judged to be the best alternative to DIPA-1-7 for cognitiveenhancement. In is possible, with the appropriate formulation, all ofthese analogs might be used as alternatives. In summary, the surprisingobservation made here was that use of these compounds, and in particularDIPA-1-7, can enhance skills requiring hand-eye coordination (e.g., insnooker) and concentration (e.g., in games of chance such as poker).

Case Study 2

A 48-year old female account executive was a busy professional at alarge financial institution. Her husband was a successful architect. Shehad two teenage children and she was constantly short of time to do herchores. At the end of the day, she was frequently physically andmentally exhausted and would fall asleep early after evening meals. Dueto recent marital difficulties, she felt tired and weary most of thetime, and her domestic and professional demeanour, in dress andetiquette, began to deteriorate. She did not suffer from any chronicphysical illness, but she was rated as having “moderate fatigue” on theBrief Fatigue Inventory (BFI) after several interviews and considered“depressed” by her physician. She volunteered to use the wipescontaining DIPA-1-7 and was instructed not to use more than one per day.After two days of use, she reported that the wipes improved her mood andinterest in external events. She was more energetic and positive. Shecompleted her assignments at work promptly and had better stamina, andshe was more combative and assertive. The people closest to her,children and work colleagues, also remarked on her improved change inattitude and personality. She continues to use the wipes on an as-neededbasis.

A 69-year old male suffered from Parkinson's disease of 12 yearsduration. He is on expert medical care and has taken a variety of drugsto help manage his disease over time. In the past several years, theprimary drugs (e.g., Sinemet®) became less effective and he was lessmobile and more housebound. In November 2009, he was implanted withelectrodes for deep brain stimulation treatment and this procedureincreased his mobility. Recently, however, in spite of carefuladjustment of his brain stimulation parameters, his Parkinsonism hasgradually returned, and he complained of constant fatigue and depressedmood. His BFI scores were in the “moderate to severe” range of fatiguelevels. He volunteered to try the wipes containing DIPA-1-7 (1 mg/mL and5 mg/mL) and was told to limit his use to one per day. The first thingthat the subject noticed, after using the wipes, was that he was able tostay awake and alert in order to watch his two favourite TV shows“House” and “Hawaii Five-0” on Monday nights (from 9 to 11 pm). He saidnormally he would have to make an extra effort to follow the dialogueand plot of “House” but would fall asleep before Hawaii Five-0 “gotgoing”. His general activity and mood improved and he was more willingto take his dog for a walk. He went to the golf range more often to dochipping and putting, but said he was still unable to turn to swinglonger clubs off the mat. His friends noticed he was in a better moodand participated more in social events. He attributes his reducedtiredness to the wipes and looks forward to its use every morning. Hesaid his appetite had improved, he longer felt depressed, and he wantedto be more active.

A 62-year old was diagnosed with hepatitis C virus (HCV) infection 10years ago and was treated with PEG-interferon and ribavarin but did notrespond because of his genetic makeup. He retired early from hisprofessional career and was relatively symptom-free except for mildfatigue which required a mandatory afternoon nap of at least two hours.However, six months ago, a 3 cm diameter hepatoma was detected bymagnetic resonance imaging on the margin of his lower right liver lobe.He was first treated by trans-arterial chemical embolization withdoxorubicin-eluting beads (TACE) and then shortly afterwards withradiofrequency ablation when it was noted that his α-fetoprotein levelswere elevated, suggesting that hepatoma cells may still be present afterTACE. These procedures resulted in moderate to severe fatigue, asevaluated by the BFI, which remained persistent even two months afterthe last treatment procedure. His initial complaint of severe pain aftersurgery was managed by the narcotic analgesic Vicodin®, but now his maincomplaint is of disturbed sleep, daytime fatigue, inability toconcentrate, and memory loss. He was prescribed the hypnotic Lunesta®,but this did not help his disturbed sleep, so he is now prescribedZolpidem®, despite the increased risks of liver damage from this drug.He volunteered to try the wipes containing DIPA-1-7 (1 mg/mL and 5mg/mL) because he was an avid reader, belonged to a book club, andwanted to keep his mind active when his mobility was physically limitedby fatigue.

After using the wipes, he commented that he was more alert and he wasbetter able to concentrate when reading. He noted that applying thewipes to a wider surface, especially on the skin of the cheekbones andorbit, enhanced the desired sensory effect. (The delivery of the sensoryagent to the neuronal receptive field is enlarged.) He noted that he hadfinished reading Kurt Vonnegut's biography but was discouraged fromtackling the biography of Steve Jobs by Walter Issacson because of itslength (more than 600 pages). After using the wipes, he finished readingthe Jobs biography in three days, and was able to remember and discussthe finer details of the book with his friends. He was especiallyintrigued by how Jobs was treated for and responded to his cancer. Hesaid his pain from surgery was not improved by use of the medicatedwipes, and he still had aches in his joints, but his mood and hisability to carry out daily activities were improved. He noted that theexceptionally long duration of action of the active ingredient in thewipes may be of use in treatment of other chronic illnesses suchnarcolepsy, neurotic and major depressions, and as an adjunct inmanaging Alzheimer's disease. He continues to use the wipes on anas-needed basis.

These studies illustrate the potential benefits of the medicated wipes,especially those containing DIPA-1-7, for countering the tiredness andfatigue of chronic illness.

Case Study 3

In another series of studies, a towelette was used for delivery insteadof a cotton wipe. The towelette consisted of a plastic wrap (weight 1.1g), a 23 cm×26 cm towel of non-woven lace (weight 3.4 to 3.5 g) and aliquid composition (14 to 15 mL) which was automatically added to andsealed off in the wrapper. Automated machinery for producing towelettesare well-known to the art. Here, the towelettes were produced by KankFactor, LLC, San Francisco (721 Commercial Street, San Francisco Calif.94108, www.3LWipes.com). These towelettes were then further treated toform either embodiments for practicing the present invention or asplacebo controls, as follows. Distilled water (as placebo controls) orDIPA-1-7 dissolved in distilled water (at a concentration of 1 to 5mg/mL) was incorporated into the towelette. The volume perself-application depended on the application site, but was about 0.3 mLto 0.5 mL for the face and brow, but could be higher if wiping of thetorso was also included.

The towelettes were stored in a refrigerator but then stored at roomtemperature for at least 1 hour before use. Effective sterilization ofthe towelette could be obtained by placement in a microwave oven for 1min [Tanaka, Y. et al. Warming and sterilizing towels by microwaveirradiation. Yonago Acta Medica 41: 83-88, 1998]. Subjects wereinstructed to hold the towelette with both hands, and bring thetowelette against the face, like how one would use a small wet facetowel, and to keep the eyes closed. The skin of the face is moistenedand medicated by this procedure. Once the subject has learned what toexpect, the subject can adjust the dosage (e.g., by dabbing), as needed,to achieve the desired anti-fatigue/anti-heat effects. After one or twotrials, individuals quickly learn how to apply the sensory agent withoutany risks of discomfort.

During an “Indian Summer” heat wave in the San Francisco Bay Area, theoutside temperature was 30 to 33° C. with a cloudless sky and an intensebright sun. The towelette, described above, was used as a substrate todeliver DIPA-1-7 to the skin of the chest and armpits of severalindividuals who complained vigorously about heat stress and discomfort.Comfortable cooling was noted for more than 3.5 hours with decreasedsweating. These Individuals were able to work normally in the heat in anoffice environment without need for additional cooling.

A 70-year old from Northern California went on a 7-day golf vacation toLas Vegas in September. He played at least one round of golf each dayand sometimes two. He did not wear a hat or use sunscreen. On the thirdday of vacation, the subject showed the classic signs and symptoms ofsunburn: redness and flushing of the facial skin, a sense of persistentwarmth, pain, and tenderness of the face, a mild degree of swellingaround the eyes, and a throbbing headache. He volunteered to try a creamcontaining 1% wt/vol DIPA-1-8 and wiped about 0.5 mL of the cream overhis cheeks and cheekbone. Surprisingly, he noted an immediate relief ofskin discomfort which lasted for at least four hours. His headache wasgone, and he said his face felt “comfortable and normal”. He used thecream on an “as needed” basis and also took measures to reduce hisexposure to direct sunlight by wearing a wide-brimmed hat and applyingcopious amounts of sunscreen products. He said that the cream would beespecially useful for the dry hot climates of Los Angeles, Phoenix andother parts of Arizona, and for Texas because also relieved the sense ofdryness and on the face gave also a feeling of “wetness” after repeateduse.

A second-year medical student in was preparing for her Boards in thesummer. During hot weather, her electricity costs increased three-fold,so that she and her roommates could not afford to turn on theair-conditioning throughout the evening hours. She said that she couldcope with the heat by using a wet towel around her neck, but the mainadverse effect of heat was disturbance of mental concentration forstudying and the difficulty in getting comfortable sleep. She agreed totry the towellette containing DIPA-1-7 and found that it gave herprolonged and refreshing cooling sensations of her face and body. Sheremarked that her skin felt fresh and cool and she was better able toconcentrate of her studies and to retain information. She also notedthat her boyfriend said that she had a fresh and energetic look aboutthe eyes, like Julia Roberts in her younger days, and that this lookmade her more attractive. She said that DIPA-1-7 may have value as acosmetic agent to enhance beauty, as well as an aid to enhanceconcentration and study in an academic situation. She also noted thatDIPA-1-7 might be useful in enhancing exercise tolerance, in the sameway that icy collars put around the neck significantly improve athleticperformance.

Case Study 4

Two scientists working in the laboratory had allergic dermatitis of thehand in response to detergents and soaps. The hands were inflamed andextremely itchy. Applications of DIPA-1-7, 20 mg/mL, with acotton-tipped applicator immediately stopped the itch and this effectlasted for at least 2 hours, and the suppression could be renewed byrepeated application. One scientist, a world-renowned dermatologist withmany publications on itch, noted that the DIPA-1-7 produced an“icy-cool” feeling on the inflamed skin and he had never encounteredsuch a compound that was so effective in stopping itch so quickly.

A pharmacologist liked to work in the garden, but the thorns frombougainvillea stems and rose bushes, and the hair from azalea leaves,irritated his skin and caused intense itch. He noted that the sensorydiscomfort on the skin could be instantly stopped by DIPA-1-6 orDIPA-1-7, applied either as a 20 mg/mL aqueous solution, or as a cream(mixed with Eucerin Moisturizing Cream). These effects could also beobtained with DIPA-1-8. He also noted that the irritation and itchcaused by insect bites could be immediately stopped by these agents.

A 40-year old suffered from penile lichen sclerosus. This is aninflammatory dermatosis of the glans penis and foreskin and, in thisparticular case, was associated with intense pruritus and dysesthesias(burning sensations). The patient, under the supervision and care of hisdermatologist, volunteered to try DIPA-1-8 on his lesion and he wassupplied with various concentrations of DIPA-1-8 dissolved in distilledwater. After self-experiment, he concluded that concentrations of 1 to1.5 mg/mL of DIPA-1-8 produced significant relief, but a concentrationof 2 mg/mL of DIPA-1-8 was too cold and uncomfortable. The solutionswere applied with cotton-tipped applicators or gauze wipes. Theadvantage of using DIPA formulations for genital skin is watersolubility. This minimizes the need for excipients and the likelihood offurther irritation. The subject suggested that an aerosolized spray mayalso be a convenient method of drug delivery.

These studies illustrate the anti-nociceptive properties of DIPA-1-7 andDIPA-1-8, especially on itching. DIPA-1-8 had a longer duration ofaction than DIPA-1-7, and may be the preferred agent for dermatologicalapplications.

Case Study 5

A 58-year old woman had early stage breast cancer, but decided to underbilateral mastectomy because of a family history of this malignancy. Sheis disease free, but has frequent bouts of hot flashes/night sweatsaveraging about 4 episodes per day. She refused suggestions of hormonereplacement therapy (HRT). She agreed to try a lotion containing 1% ofDIPA-1-7. This lotion was applied on the skin at the base of her neckand on the centre of her chest before going to sleep at night. If shewoke up at night, the application was repeated. She said the lotion feltcool, but it was not effective on her hot flashes. After discussion withher physician she decided to try a cherry-flavored syrup [Humco Corp.,Austin, Tex.] containing 8 mg/mL of DIPA-1-9. This syrup was deliveredfrom a 15 mL squeeze bottle and administered to the back of the mouth,on the tongue, when the subject felt the possible onset of a “hotflash”. The delivered volume per dose averaged 0.5 mL. The DIPA-1-9syrup produced a rapid cooling in her throat and then the center of herchest but was not felt to be aversive. Surprisingly, the frequency andintensity of hot flashes diminished and the subject was convinced thatthe cooling effects of the DIPA-1-9 syrup on her pharyngeal andesophageal linings were effective in controlling her symptoms.

Case Study 6

Three subjects decided to systemically compare DIPA-1-6, DIPA-1-7,DIPA-1-8, and DIPA-1-9 for their sensory effects on the ocular surface.Each compound was prepared at 1 mg/mL in distilled water. A cottontipped applicator of a specific size (Puritan 803-PCL) consisting of a55 to 75 mg ball of cotton wound around the tip of a three inchpolystyrene rod was dipped into the solution. The tip was then applied,with the eyelids closed, to the lower aspect of the upper eyelid, ontothe eyelashes, with two lateral to medial wiping motions. The subjectswere then instructed to blink. By blinking, the solution is then evenlydistributed over the pre-corneal film. This “swab” delivery methodoff-loaded a total of ˜35 μL of liquid onto the surface of both eyes.DIPA-1-6 caused significant stinging and discomfort and was thereforenot further studied. DIPA-1-7 and DIPA-1-8 produced strong andrefreshing cooling, which counter-acted eye irritation, and increasedcognitive functions. For example, subjects felt they could focus ondistant objects and enjoy the view. They felt mentally alert andrefreshed. But, with both DIPA-1-7 and DIPA-1-8, there was a smallresidue left on the eyelid; subsequently using a towel to wash the facecan cause eye irritation. Surprisingly, DIPA-1-9 did not produce any eyeirritation when wiped over the eyelid, nor did it leave a residue. Italso produced refreshing cooling, but not with the same intensity asDIPA-1-7 or DIPA-1-8. On the other hand, DIPA-1-9 has ideal propertiesfor the treatment of ocular discomfort, e.g., discomfort caused by eyestrain; eye fatigue; eye surgery; an airborne irritant or pollutant thatinteracts with the eye surface; extended wear of contact lenses;excessive exposure to the sun; conjunctivitis; or the dry eyes syndrome.

Case Study 7

The surprising lack of irritation on the ocular surface seen withDIPA-1-9 in Case Study 6 prompted its investigation on another site: thesingle cell layer of the respiratory epithelium in the nasal cavity. Asolution of DIPA-1-9, 2 mg/mL in isotonic saline, was placed in a 15 mLplastic squeeze bottle with a fine eye-dropper tip. The solution wasinstilled at about 3 to 4 drops into the nares of 10 subjects withvarying degrees of nasal congestion from the common cold and fromallergic rhinitis. The results were dramatic and amazing. Subjects feltinstant clearing of the nasal passages and this effect lasted for 3 to 6hours! Thus, a new mechanism for the treatment of nasal congestion wasdiscovered.

Keh et al. [The menthol and cold sensation receptor TRPM8 in normalhuman nasal mucosa and rhinitis. Rhinology 49, 453-7 (2011)] havedetected TRPM8 immunoreactivity in human nasal mucosa, closelyassociated with nerve fibers and blood vessels. The immunoreactive TRPM8proteins in the nasal mucosa were not increased in patients withrhinitis. Keh et al. [2011] suggested that TRPM8 antagonists might havevalue in rhinitis. We were able to confirm the presence of TRPM8 nervefibers in the nasal epithelium using green fluorescent protein labelingof TRPM8 in genetically modified mice [studies by Li FengXian atWashington University, St. Louis, Mo.].

But we propose here a view that is opposite to that of Keh et al.;namely, TRPM8 agonists have beneficial effects in the nasal discomfortcaused by rhinitis and not antagonists. In science there is often timesconfusion when one group says that the agonist will work, and anothergroup advocates the antagonist. The data of this discovery clearly favorthe agonists and not the antagonists. DIPA-1-9 is a preferred embodimentof this drug action on the nasal membranes because it is long-acting andnot irritating. Related analogs, DIPA-1-7, DIPA-1-8, 2-6 and 2-7 werealso tested on nasal membranes but caused rhinorrhea by stimulating thecold receptors on the nostril skin.

Case Study 8

A 2-year old female West Highland Terrier developed, during the summer,an itching condition which led to continued scratching of the ears andunderbelly. The veterinarian diagnosed the behavior as canine atopy andprescribed oral antihistamines. These drugs did not control theprogression of the itching and patches of raw skin, with hair loss,occurred at the base of the tail and on the hind limbs. A topicalanti-inflammatory steroid, triamcinolone, provided limited success butthe dog still looked miserable. Surprisingly, application of DIPA-1-7cream (1% wt/vol) to the inflamed skin sites immediately reducedscratching and the skin sites begin to heal. It was clear from the dog'sbehavior that the itching was reduced in severity. Further curtailmentof the dog's access to the outdoors and control of possible exposure tofleas and dust mites resulted in a successful control of the dog's skindisorder.

Case Study 9

The eyes are extremely sensitive to injury and the symptoms of injuryinclude blurring of vision, itching, irritation, burning sensations,sensations of a foreign object, and pain. The ability of DIPA-1-9 toproduce prolonged cooling without residual discomfort suggested that itmight be useful in the treatment of the “dry eyes syndrome”, a widelyprevalent condition in the general population. Prof K C Yoon, theleading ophthalmologist for “dry eyes syndrome” [DES] in Korea conducteda clinical trial of DIPA-1-9 in normal and in patients diagnosed withDES. For this DIPA-1-9 study, there were 12 normal subjects and 15 DESpatients. The characteristics of the study population are shown in Table11. The trial was approved by the Institutional Review Board of ChonnamUniversity, Gwangyu, Korea.

Ten drops of DIPA-1-9, 2 mg/mL in saline, was placed on a cotton gauzepad and wiped onto the upper eyelids of subjects, with the eyes closed.Change in symptoms of ocular surface cooling sensation were obtained byquestionnaire using a visual analog scale scores (0 to 10) at every 5min interval. The tear film break-up time (BUT) was measured at 10 minintervals, and the Schirmer test I (without anesthesia) was measured at20 min intervals, the ocular surface epithelial damage score(keratoepitheliopathy) was recorded using the National Eye Institute(NEI) system. Corneal sensitivity to a microfilament was measured usingCochet-Bonnet esthesiometry.

After application the onset of coolness reached a peak score of 6 within5 min and steadily decreased thereafter. The average duration ofcoolness was 47 min and there was no difference between the two subjectgroups. The tear BUT and the Schirmer test 1 scores were significantlyimproved in the DES group, but there were no significant changes in thescores of “normal” subjects. The effects on BUT and Schirmer lasted for˜30 min after application of DIPAP-1-9. There were no changes in cornealsensitivity to mechanical stimulation.

In the opinion of Prof Yoon the symptomatic relief seen in the DESpatients receiving DIPA-1-9 is quantitatively better than the resultsseen in his laboratory with approved drugs for DES; namely, cyclosporine0.5% and diquasol, a P2Y2 agonist. Further studies, with a larger testgroup and a 4-week regimen of treatment are in progress and the resultswere publicly communicated at the Association for Vision andOpthlamology (ARVO) meeting, Seattle, Wash. May 1-5, 2016. The Abstractof this presentation is shown below:

KC Yoon et al. Efficacy of topical application of a transient receptorpotential melastatin 8 agonist in patients with dry eye. Program No.2877, Poster A0086. Purpose: To investigate the efficacy of topicaladministration of a transient receptor potential melastatin 8 (TRPM8)agonist in patients with dry eye disease. Methods: In this randomizedprospective double-masked study, 60 subjects (60 dyes) with mild tomoderate dry eye disease were recruited and allocated to two groups: aTRPM8 agonist (1-diisopropylphosphorylnonane; group A) or vehicle (groupB). Study medications were topically applied 4 times a day for 2 weeksto the upper eyelids using a single unit applicator containing the TRPM8agonist, 2 mg/mL, in water, or vehicle. Tear break-up time (TBUT),Schirmer score, and keratoepitheliopathy (KEP) score were analyzedbefore and at 1 and 2 weeks thereafter. In addition, dry eye symptomsassessed by the visual analogue scale (VAS), ocular surface diseaseindex (OSDI), and grade of computer vision syndrome (CVS) were evaluatedbefore and after application. Results: The changes in TBUT and KEP werenot significant in both groups. The Schirmer score increased in group Aat 1 and 2 weeks when compared to baseline and when compared to group B(P<0.05). The changes in ocular symptom scores, assessed by threequestionnaires, showed that the VAS and total OSDI scores were improvedat 2 week and the CVS score at 1 and 2 weeks in group A compared togroup B (P<0.05). No significant adverse effect such as ocular pain,irritation, or discomfort was reported from both groups during the studyperiod. Conclusions: Daily topical application of the TRPM8 agonist onthe eyelid skin was safe and effective in improving tear secretion andocular symptoms in patients with dry eye. The agent is a promisingcandidate for further exploration of TRPM8 function on the ocularsurface and for use in the relief of dry eye discomfort. Clinical TrialRegistration: ISRCTN12259367.

A key aspect to the success of the Yoon et al study was to apply theactive ingredient onto the eyelid receptors with a wipe. The testsolution was wiped onto the upper eyelids and touching the lashes. Onewould expect the eyelash shaft to serves as a wick to distribute theaqueous solution across the eyelid margins and merge with the precornealfilm which is also aqueous. The keratinized epithelial cushion, calledthe eyelid wiper, would help push the solution towards the drainagepuncta at the corner of the eye. Using these methods, comfortable andprolonged ocular surface cooling was achieved without discomfort.

Traditional methods are to apply ocular drugs using eye drops. Theadvantages of the pre-medicated wipe delivery method over eye drops areseveral-fold. The wiping volume is small, a total ˜44 μL to both eyes,and contact with the corneal surface is minimized. The pre-medicatedwipe packages are sterile and disposable. The Intricacies of hand-eyecoordination, angulation of the head, and avoiding accidental contact ofthe eye drops bottle tip to the eye surface, are avoided. There are nobolus effects on the corneal surface of eye drops and, hence, noirritation and discomfort is observed. The even distribution of theactive ingredient to the eyelid receptors is accomplished. In practice,the dispersion of the drug solution by the wipe method on the targetsite is radically different from the drug dispersion obtained with aneye drop and is based on the histological studies identifying the targetreceptors on the eyelids.

In summary, the unusual properties of DIPA-1-9 as a non-irritating,long-acting agent on non-keratinizing membranes of the ocular surface,nasal membranes, pharyngeal, and esophageal surfaces have been revealed.Studies described herein indicate that DIPA-1-9 can be used to treatocular discomfort, nasal congestion, pharyngeal and esophagealdiscomforts, with a mode of drug action that has not been previouslyobserved or recognized.

REFERENCES

A number of publications are cited herein in order to more fullydescribe and disclose the discovery and the state of the art to whichthe discovery pertains. Each of these publications is incorporatedherein by reference in its entirety into the present disclosure

The invention claimed is:
 1. A method of treating fatigue in a humansubject experiencing fatigue, comprising: topically applying atherapeutic composition to the human subject or instructing a user totopically apply the composition to the human subject wherein thecomposition has an amount of a compound with Formula 1 therein

wherein R is n-heptyl, n-octyl and/or n-nonyl and wherein the appliedamount of Formula 1 compound in the composition is from 0.01 to 10 mgper unit dose.
 2. The method as in claim 1 wherein the compound is1-diisopropyl-phosphinoyl-heptane [DIPA-1-7].
 3. The method as in claim1 wherein the compound is 1-diisopropyl-phosphinoyl-octane [DIPA-1-8].4. The method as in claim 1 wherein the compound is1-diisopropyl-phosphinoyl-nonane [DIPA-1-9].
 5. The method as in claim 1wherein the composition is administered topically via a swab, wipe, pad,or towellette.
 6. The method as in claim 1 wherein the composition is inthe form of a paste, gel, lotion, cream, ointment, spray or aerosol. 7.The method as in claim 1 wherein the composition comprises water,isotonic saline, or an aqueous buffered solution.
 8. The method as inclaim 1, wherein the fatigue is caused by chronic illness, cancer orcancer-related treatment, ageing, an impairment of vision, aneurological dysfunction, or a psychological dysfunction.
 9. The methodas in claim 1, wherein the fatigue is fatigue caused by anxiety,depression, heat stress, cognitive dysfunction, excessive physicalexertion, or excessive mental exertion.
 10. The method as in claim 1,wherein the fatigue is associated with a decreased ability to think, toconcentrate, to study, or to perform work.
 11. The method as in claim 1,wherein the treatment improves hand-eye coordination in a sport, andimproves performance in a game of chance or of mental skills.